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High rates of embryonic loss, yet high incidence of multiple births in human ART: is this paradoxical?
Abstract
Humans have low natural fecundity, as the probability of establishing a viable conception in any one menstrual cycle is 20-25% for a healthy, fertile couple. There are numerous underlying causes for this low rate of human fertility, not the least of which are intrinsic abnormalities within the oocyte and/or embryo, which likely account for greater than 50% of failed conceptions. During assisted reproduction technology (ART) interventions, controlled ovarian stimulation is used to obtain several oocytes in attempts to increase the likelihood of having at least one developmentally competent embryo available for transfer. However, current techniques for identifying the competent embryo(s) are by no means perfect. These limitations, coupled with pressures to maximize the chance of pregnancy, typically result in the transfer of multiple embryos. Not surprisingly, this practice has resulted in an unacceptably high rate of multiple pregnancies arising from ART. During the last few years, concerted efforts have focused on reducing these rates. Programs for ART are developing patient-specific policies, restricting the number of embryos to transfer. In addition, strategies are being adopted to improve the accuracy for selecting viable embryos for transfer. One such strategy involves further refinement of morphological criteria associated with improved viability by considering, for example, pronuclei disposition, nucleolar organization, and identification of the fast-cleaving embryos with only mononucleate blastomeres. Another strategy employs pre-implantation genetic diagnosis (PGD) whereby a biopsied blastomere is tested for ploidy using fluorescence in situ hybridization (FISH). A final strategy involves extending the duration of culture to the blastocyst stage, thereby allowing self-selection of those embryos capable of proceeding to blastulation and exclusion of those less viable embryos that succumb to developmental arrest. Together, these strategies are enabling fewer embryos of higher quality to be transferred. Accordingly, the overall pregnancy rate from ART continues to increase, while the rate of triplet and higher order multiple births continues to decline. Nevertheless, the high incidence of intrinsic developmental anomalies in human oocytes inevitably will continue to result in a high degree of embryonic loss in ART.
... According to Coughlan et al., the term "implantation failure" refers to two different types of cases, those in whom there has never been evidence of implantation (no detectable HCG production) and those who have evidence of implantation (detectable HCG production) but it did not proceed to beyond the formation of a gestational sac visible on ultrasonography (1). It is rather doubtful whether such an event should be called a pathological event because it has been reported that spontaneous pregnancy is achieved in only about 25-40% of healthy fertile women during the first cycle of intended pregnancy (2,3). Because of the importance of this problem and its correct definition, a whole section of this review was dedicated to the subject, presenting viewpoints published in the literature so far. ...
... After widespread application of assisted reproduction technologies (ART) and in particular IVF/ ICSI, a novel pathophysiological state was recognized which was characterized by numerous failures to achieve pregnancy after embryo transfer (ET) and it was designated as recurrent implantation failure (RIF). There is no universally accepted definition despite many publications on this topic (2,(5)(6)(7)(8). Collective data from papers reporting implantation rates in different ART clinics strongly suggest that the maximum implantation rate is between 40% and 60% (7). ...
... Currently, selective single embryo transfers are performed in many countries and some authors are inclined to define RIF as a failure to achieve pregnancy after 3 embryo transfers with good quality embryos (6). The latest proposed definition of RIF from Coughlan et al. includes not only the number of embryos and ET-procedures but also the age of females (2). ...
The success rate of reproductive treatment methods depends on many different factors. The most important and discussed ones in the literature are maternal age, the causes of infertility, the ovarian response to stimulation, the influence of the male factor and sperm quality, embryo quality and the various uterine pathologies. Some couples fail repeatedly after transferring good quality embryos without any obvious reason and this becomes a major continuing problem after IVF/ICSI procedures. It can be speculated that in these couples, insufficiency of the endometrium might be a possible reason for implantation failure. This review article summarized current literature describing the consecutive endomertial procedures involved in successful embryo implantation. It is believed that efforts to align criteria for definition of recurrent implantation failure (RIF) and attempts to classify different RIF types would develop guidelines for treatment procedures which would result in an increase in patients' opportunities to conceive.
... In spite of all these progressions, both in the technologies and knowledge regarding embryo development, the success rates for IUI, IVF or ICSI remains reasonably low all over the world. The implantation failure rate is still pretty high in spite of choosing best quality embryos according to their morphology for transfer (Racowsky, 2002).. Various studies pointed out that the clinical pregnancy rate after transfer of single embryos is 40-60% (Jain et al., 2004;Leniaud et al., 2008;Stillman et al., 2009). In some IVF clinics, more than one embryo is transferred to increase the chances of pregnancy which results in multiple pregnancy and other associated complications like low-birth weight (LBW) and prematurity (Racowsky, 2002). ...
... The implantation failure rate is still pretty high in spite of choosing best quality embryos according to their morphology for transfer (Racowsky, 2002).. Various studies pointed out that the clinical pregnancy rate after transfer of single embryos is 40-60% (Jain et al., 2004;Leniaud et al., 2008;Stillman et al., 2009). In some IVF clinics, more than one embryo is transferred to increase the chances of pregnancy which results in multiple pregnancy and other associated complications like low-birth weight (LBW) and prematurity (Racowsky, 2002). Moreover, the methods of ART are expensive, time consuming and the couples has to go through a stage of rigorous stress (Murphy, 2007). ...
Advanced technologies have steadily been established for conserving the potential of biological parenthood in couples who are at risk of losing their fertility due to suffering from benign, malignant cancers as well as other genetic diseases. Methods for fertility preservation differ significantly, depending mainly on the patient’s age and the disease they are suffering from. Cryopreservation of gametes has been suggested to be a putative novel approach to preserving fertility in women and men who are at risk of losing fertility. However, the effects of the freezing/thawing process are not fully understood and ways to assess the tissue’s potential to perform normally after thawing, are an important research goal.
MicroRNAs are evolutionarily conserved, single-stranded, non coding RNA molecules, consist of 18–22 nucleotides. They are important intercellular signalling molecules, known to have major roles in post transcriptional gene regulation and protein synthesis. In many cells, specific microRNAs are expressed differentially in disease states compared to normal tissue. Recent reports have identified microRNAs during the process of folliculogenesis and spermatogenesis which may be able to modulate reproduction. Over 200 microRNAs have been reported in human follicular fluid and using bioinformatics analysis, microRNA expression in granulosa cells and follicular fluid and its correlation with endocrine, reproductive and metabolic functions have been studied. During spermatogenesis they are also expressed in a precise manner, therefore taking part in every step of male germ cell development. The lack of tight regulation of these processes by microRNAs can cause disruptions and prevent certain stages of development occurring within the correct time frame during folliculogenesis or spermatogenesis, and it can lead to abnormalities in oocyte or sperm development.
In this study, we investigated the possible association between microRNA expression pattern and cryopreservation that could help in the assessment of ovarian tissue damage and oocyte/sperm quality post freeze-thaw cycles. We have selected five target microRNAs (24, 193b, 320A, Let7-b and 34C), as these microRNAs have been shown to play an important role in regulation of folliculogenesis and spermatogenesis in a variety of species.
Chapter 3 investigated the possible interplay between steroid production and microRNAs’ expression pattern that could help in the assessment of tissue damage post freeze-thaw cycles. Ovine ovarian cortical tissues were cultured in rotatory cell culture system with or without cryopreservation, spent culture media from the system was analysed for steroid production (oestradiol and progesterone) and microRNA expression profile (24, 193b and 320A). The number of primordial, transitory, primary, secondary and antral follicles were analysed between fresh, fresh-cultured and cryopreserved-cultured ovarian cortical tissues. The findings confirmed that, downregulation of miRNA-193b and miRNA- 320A together with upregulation of miRNA-24 could have a synergistic role in cell apoptosis, and consequently leading to reduced oestradiol and progesterone production, which can be further explored as novel non-invasive markers of cell damage following cryopreservation.
Chapter 4 investigated the expression pattern of three microRNAs (24, 193b and 320A) in spent culture media supplemented with or without Insulin-Transferrin-Selenium as indicators of oocyte quality; post maturation and post vitrification. Ovine oocytes were matured in media supplemented with or without ITS. Following maturation, oocyte cumulus expansion was measured, and viable oocytes were then vitrified and tested again for viability after warming. The changes in expression pattern of microRNAs’ in spent culture media supplemented with or without ITS was investigated, both post- maturation and post- vitrification, and the differential expression was correlated with cumulus expansion, oocyte maturation and oocyte viability. The findings confirmed that, oocytes matured in media supplemented with ITS showed a significant increase in cumulus expansion and nuclear maturation, and viability following vitrification. Downregulated miRNA- 24 and 193B and upregulated 320A might be associated with improved oocyte maturation, viability and cumulus expansion, which could be boosted by the presence of ITS supplementation. These miRNAs can further be explored as markers of oocyte quality following maturation and vitrification.
Chapter 5 investigated the expression pattern of five microRNAs (24, 193b, 320A, Let-7b and 34C) in human semen samples as marker of sperm quality, pre and post density gradient centrifugation. The expression pattern of these microRNAs was correlated with semen parameters (concentration, progressive motility and velocity). The findings confirmed that, the effect of DGC in samples was a positive one based on the expression levels of target miRNAs. Also, negative correlation of microRNA-193b and positive correlation of microRNA-34C with velocity along with increased expression of microRNA-34C following DGC can possibly be further re-enforced utilised as a marker for fertility.
Chapter 6 investigated the possible association between sperm cryopreservation and semen parameters (concentration, progressive motility and velocity)/miRNAs’ expression pattern that could help in the assessment of sperm quality post freeze-thaw cycles. Semen parameters and miRNAs’ (24, 193b, 320A, Let-7b and 34C) expression profile was evaluated in human spermatozoa subjected to cryopreservation (controlled rate freezing and vapour cooling) using 2 different types of cryoprotectants (Test-yolk buffer and Quinns advantage sperm freezing media). Both the freezing methods (controlled rate freezing and vapour cooling) as well as CPAs (Test-yolk buffer and Quinns advantage sperm freezing media) were comparable in maintaining sperm concentration, PM% and velocity. Nonetheless, based on the findings of microRNAs’, Quinns advantage sperm freezing media seems to be superior to Test-yolk buffer using both the freezing methods (controlled rate freezing and vapour cooling.
In conclusion, down-regulation of microRNA-193b and microRNA-320A along with up-regulation of microRNA-24 for cryopreserved then cultured ovarian cortical tissue might have a synergistic role in cell apoptosis and consequently reduced oestradiol and progesterone production. Moreover, downregulated microRNA-24 and microRNA-193B and upregulated microRNA-320A plays an important role in oocyte maturation, viability and cumulus expansion in in presence of ITS. These microRNAs’ (24, 193b and 320A) could be further explored as novel markers of cell injuries and/or damage following cryopreservation for ovarian cortical tissue and oocytes. As for the sperm, the negative and positive correlation of microRNA-193b and microRNA-34C respectively with velocity can possibly be further utilised as a marker for sperm quality following preparation and cryopreservation. Both tested sperm freezing methods were comparable in maintaining sperm concentration, progressive motility and microRNA expression, nonetheless, supplementation of egg yolk resulted in poorer outcomes.
... Moreover, abnormalities are more frequent not only in arrested embryos, but also in embryos with slow or accelerated cleavage, emphasizing the importance of the time of ®rst cleavage for correct development (Yadav et al., 1993; Magli et al., 1998 Magli et al., , 2001). A clear relationship between the time of ®rst cleavage and developmental potential has been previously demonstrated for both bovine and human oocytes, with the earliest cleaving oocytes being more likely to develop to the blastocyst stage than those that cleave late (Plante et al., 1994; Lonergan et al., 1999 Lonergan et al., , 2000 Ward et al., 2001; Fenwick et al., 2002; Racowsky, 2002; Lequarre et al., 2003). It was also observed that timing of ®rst cleavage is associated with different stability of speci®c transcripts necessary for correct development (Brevini et al., 2002) and differential maternal mRNA expression (Fair et al., 2004). ...
... A association between time of ®rst cleavage and the rate of early embryonic arrest was observed, with late cleaving embryos more likely to arrest at the 2±4-cell stage than early cleaving embryos. This supports previous observations in bovine (Yadav et al., 1993; Plante et al., 1994; Lonergan et al., 1999 Lonergan et al., , 2000 Ward et al., 2001; Lequarre et al., 2003) and human embryos (Magli et al., 1998Magli et al., , 2001 Fenwick et al., 2002; Racowsky, 2002 ) that demonstrated increased developmental capability of early cleaving embryos. Furthermore, we hypothesized that these arrested embryos enter a `senescence-like state', which in fetal bovine ®broblasts is associated at the molecular level by the increased abundance of p53 and p66 shc (L.A.Favetta et al., unpublished data). ...
High embryo loss occurs in the first week of bovine embryo development, with a high percentage of embryonic arrest. We hypothesized that arrested embryos enter a 'senescence-like state' and that both the cell cycle regulatory protein p53 and the stress-related protein p66shc, which are involved in the onset of senescence in somatic cells, are responsible for this early embryonic arrest. In our in vitro production system, 13.5 6 6 0.5% of embryos arrest at the 2-4-cell stage. First cleavage occurs between 26 and 48 h post insemination (hpi), with early cleaving embryos showing only 0.6 6 6 0.3% arrest, with later cleaving embryos exhibiting up to 14.2 6 6 0.9% arrest. We compared 2-4-cell embryos collected at 28 hpi with those arrested at the 2-4- cell stage collected at day 8 post insemination. Quantification by real-time PCR and by semi-quantitative immunofluorescence showed significantly higher p66shc mRNA and protein levels in both arrested and late cleaving embryos versus 28 hpi embryos. By comparison, no significant changes in p53 mRNA, protein and phosphorylation levels were detected. Taken together, these results demonstrate that embryonic developmental potential is related to the time of first cleavage and that p66shc, but not p53,
... A substantial proportion of human embryos are lost before their implantation in the uterus and therefore too early to be detected as pregnancies even by elevated HCG levels. It has been calculated that ∼47% of in vivo-fertilized human embryos are lost in the first 2 weeks (Leridon, 1973;Racowsky, 2002). It is most likely that suboptimal oocyte quality is a major source of this loss. ...
... It is most likely that suboptimal oocyte quality is a major source of this loss. Further, although birth rates for assisted reproduction techniques have now reached approximately the levels expected for in vivo (∼25-35% per cycle) (Racowsky, 2002;Macklon et al., 2002); this rate is achieved only through the practice of multiple embryo transfer (usually two or three). ...
A nonhuman primate model was applied to investigate the relationships between variations in the organization of microtubules, microfilaments, and chromatin in metaphase I and metaphase II oocytes. Marmoset oocytes were subjected to in vitro maturation and coincubation with sperm. Oocytes which failed to cleave were investigated for chromatin, tubulin, and actin using Hoechst 33258, fluorescein isothiocyanate (FITC)-labeled alpha-tubulin antibody and rhodamine-labeled phalloidin, respectively. Spindles were categorized according to size, shape and microtubule organization: normal, large, multipolar, disorganized, absent spindle, and spindles with broad poles. Actin caps were categorized as: normal, small, split, and disorganized. Chromosomal condensation and alignment were described as normal or abnormal. Improper chromosomal condensation was associated with both abnormal microfilament and microtubule arrangement. This was further associated with abnormal actin organization, disorientation and late stabilization of microtubules, but not related to abnormal organization of spindle poles. Chromosomal misalignment was associated with disorientation and late stabilization of tubulin, but not to broad spindle pole. Additionally, abnormal actin polarization appeared not to be related to abnormal spindle poles. The model system presented in this study could be used as an experimental platform for studying the contribution of different factors to the exactness of late meiotic events in primate oocytes. The present study provides basic information on spindle, chromosome, and actin normal and abnormal organization, which can be observed in in vitro matured, but failed to cleave primate oocytes.
... This force is not only active after implantation and during embryo development, but surprisingly it works controlling the implantation process very powerfully. The rate of unsuccessful outcomes rises the 80% of total pregnancies if we consider miscarriage from the first steps of pregnancy after fecundation (Apanius et al. [1997]; Gilbert et al. [1998]; Racowsky [2002]). In mice, a particular MHC-driven selective behavior, the "Bruce effect", has been observed: a pregnant female, which has the chance to mate with a second partner that differs at MHC loci, aborts in favour of the more MHC-different male (Yamazaki et al. [1983a]). ...
... In the first steps of implantation there is probably a sequential and fine regulation in which a proinflammatory moment is followed by an anti-inflammatory environment. In fact, modern tests suggest that the total miscarriage rate is up to 80%, including embryos lost in the implantation phase (Racowsky [2002]). Thus, we might be expected to have evolved some genetic adaptation that protect us against miscarriage, while exposing us to new postnatal curses. ...
The Major Histocompatibility Complex (MHC) is considered a system completely defined and only connected with the immune response. However, in addition to the well-known correlation between MHC and the non-self recognition, the MHC region controls a lot of other functions: the recognition of genetic individuality in social relationships, the mate choice and the feto-maternal interplay. Starting from protocordates, the first MHC function was the individual self-identification inside a group, but then it turned into an inter-individual recognition system, which could transmit information about the MHC genotypes. In mammals, the MHC system is functionally and physically linked to the olfactory receptors: when smelling each other, we are able to make a direct genetic analysis through the nose. The MHC individual genetic recognition system plays a fundamental role, both in mate choice and in foeto-maternal selection, from the very start of implantation. All these data suggest that the MHC polymorphism is driven not only by pathogen selection, but also by sexual reproductive-mechanisms. Questions remain about the relative involvement of these two selective forces in MHC evolution.
... In all mammalian species, the greatest constraint to reproductive performance is embryonic mortality, which counts 20-30% of embryos in most cases; however, the true rate of early pregnancy loss is close to 50% due to the high number of pregnancies that are not recognized in the first 2 to 4 weeks after conception (Bazer and First 1983;Patel and Lessey 2011;Racowsky 2002). Intrauterine growth restriction (IUGR), defined as impaired growth and development of fetuses and their organs during pregnancy, is also commonly observed in mammals, and in turn leads to "runt" offspring with poor survival in the neonatal period of life (McMillen and Robinson 2005;Wu et al. 2006;Wang et al. 2010Wang et al. , 2013Wang et al. , 2018. ...
Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr2) isolated from Day-12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via activation of mechanistic target of rapamycin (MTOR) cell signaling pathway in pTr2 cells. Porcine ADM at 10–7 M stimulated (P < 0.05) pTr2 cell proliferation, migration and adhesion by 1.4-, 1.5- and 1.2-folds, respectively. These ADM-induced effects were abrogated (P < 0.05) by siRNA-mediated knockdown of ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL), as well as by rapamycin, the inhibitor of MTOR. Using siRNA-mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph acts on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation, migration and attachment of conceptuses.
... 37 The blastulation rate represents the synthesis of both the clinician's and embryologist's involvement and the blastulation rate relates to the ART chances of success. 39 Among the selected morphokinetic parameters, the morula time seemed predictive of embryo implantation and this is consistent with the fact that this parameter has been shown to be predictive of live birth. 50 Similarly, late blastulation has been shown to be correlated with a drop in the chances of implantation, 51 this is why this morphokinetic parameter (tB), should be included. ...
... 37 The blastulation rate represents the synthesis of both the clinician's and embryologist's involvement and the blastulation rate relates to the ART chances of success. 39 Among the selected morphokinetic parameters, the morula time seemed predictive of embryo implantation and this is consistent with the fact that this parameter has been shown to be predictive of live birth. 50 Similarly, late blastulation has been shown to be correlated with a drop in the chances of implantation, 51 this is why this morphokinetic parameter (tB), should be included. ...
Purpose:
The purpose of this work was to construct shallow neural networks (SNN) using time-lapse technology (TLT) from morphokinetic parameters coupled to assisted reproductive technology (ART) parameters in order to assist the choice of embryo(s) to be transferred with the highest probability of achieving a live birth (LB).
Methods:
A retrospective observational single-center study was performed, 654 cycles were included. Three SNN: multilayers perceptron (MLP), simple recurrent neuronal network (simple RNN) and long short term memory RNN (LSTM-RNN) were trained with K-fold cross-validation to avoid sampling bias. The predictive power of SNNs was measured using performance scores as AUC (area under curve), accuracy, precision, Recall and F1 score.
Results:
In the training data group, MLP and simple RNN provide the best performance scores; however, all AUCs were above 0.8. In the validating data group, all networks were equivalent with no performance scores difference and all AUC values were above 0.8.
Conclusion:
Coupling morphokinetic parameters with ART parameters allows to SNNs to predict the probability of LB, and all SNNs seems to be efficient according to the performance scores. An automatic time recognition system coupled to one of these SNNs could allow a complete automation to choose the blastocyst(s) to be transferred.
... Human embryo implantation is an inherently inefficient process. Only around 25% of blastocysts implant into the uterus, representing a major hurdle to natural conception [38][39][40] and ratelimiting step in assisted reproductive technologies 41,42 . In 2% of pregnancies, the embryo implants outside of the endometrium, often into the fallopian tubes, resulting in an ectopic pregnancy 43 . ...
The uterus is the organ for embryo implantation and fetal development. Most current models of the uterus are centred around capturing its function during later stages of pregnancy to increase the survival in pre-term births. However, in vitro models focusing on the uterine tissue itself would allow modelling of pathologies including endometriosis and uterine cancers, and open new avenues to investigate embryo implantation and human development. Motivated by these key questions, we discuss how stem cell-based uteri may be engineered from constituent cell parts, either as advanced self-organising cultures, or by controlled assembly through microfluidic and print-based technologies.
... 1,2 In humans, the natural conception rate per menstrual cycle is approximately 30%, and 75% of all pregnancy failures are believed to be due to embryo implantation failure. 3,4 The pregnancy rate for in vitro fertilized embryos is 30%-40% 5 due to embryo implantation failure. 6 In addition, more than 90% of embryos utilized in nuclear transfer technology die during the pregnancy period. ...
Objectives
Early pregnancy loss is a major clinical concern in animal and human reproduction, which is largely influenced by embryo implantation. The importance of methionine for embryo implantation is widely neglected.
Materials and methods
We performed a series of experiments with primiparous rats fed diets containing different levels of methionine during early pregnancy to investigate the role of methionine in embryonic implantation and pregnancy outcomes, and used them to perform in vivo metabolic assessments and in vitro uterine explant culture. In addition, through transcriptome analysis and silencing the expression of cystathionine β‐synthase (CBS, the key enzyme in transsulfuration pathway) and cell adhesion assay, we measured signalling within Ishikawa, pTr and JAR cells.
Results
We determined the relevance and underlying mechanism of methionine on embryo implantation. We showed that methionine deprivation sharply decreased embryo implantation sites, expression of CBS and transsulfuration pathway end products, which were reversed by maternal methionine supplementation during early pregnancy. Moreover, we found CBS improved methionine‐mediated cell proliferation and DNA synthesis by CBS inhibition or interference. In addition, transcriptome analysis also revealed that CBS influenced the signalling pathway‐associated cell proliferation and DNA synthesis, as well as a correlation between CBS and methionine adenosyltransferase 2A (MAT2A), implying that MAT2A was possibly involved in cell proliferation and DNA synthesis. Further analysis revealed that MAT2A influenced S‐adenosylmethionine receptor SAMTOR expression, and SAMTOR activated mTORC1 and its downstream S6K1 and CAD, ultimately enhancing DNA synthesis in the embryo and uterus.
Conclusions
Taken together, these studies demonstrate that CBS and MAT2A improve methionine‐mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation.
... Currently, it is estimated that more than 9 million babies have been born worldwide since the first IVF baby was born in 1978, and IVF contributes to 1-5% of all newborns in developed countries [1]. Although the great majority of IVF-conceived offspring are in good health, increasing epidemiologic analyses in humans and laboratory studies in animals show that IVF is associated with various short-or long-term consequences, including pregnancy complications, preterm birth, low birth weight, birth defects [2][3][4][5][6], as well as higher disease risks in later life, such as heart disease, diabetes, and hypertension [7][8][9][10][11]. ...
Well-orchestrated epigenetic modifications during early development are essential for embryonic survival and postnatal growth. Erroneous epigenetic modifications due to environmental perturbations such as manipulation and culture of embryos during in vitro fertilization (IVF) are linked to various short- or long-term consequences. Among these, DNA methylation defects are of great concern. Despite the critical role of DNA methylation in determining embryonic development potential, the mechanisms underlying IVF-associated DNA methylation defects, however, remains largely elusive. We reported herein that repression of fibroblast growth factor (FGF) signaling as the main reason for IVF-associated DNA methylation defects. Comparative methylome analysis by postimplantation stage suggested that IVF mouse embryos undergo impaired de novo DNA methylation during implantation stage. Further analyses indicated that Dnmt3b, the main de novo DNA methyltransferase, was consistently inhibited during the transition from the blastocyst to postimplantation stage (Embryonic day 7.5, E7.5). Using blastocysts and embryonic stem cells (ESCs) as the model, we showed repression of FGF signaling is responsible for Dnmt3b inhibition and global hypomethylation during early development, and MEK/ERK-SP1 pathway plays an essential mediating role in FGF signaling-induced transcriptional activation of Dnmt3b. Supplementation of FGF2, which was exclusively produced in the maternal oviduct, into embryo culture medium significantly rescued Dnmt3b inhibition. Our study, using mouse embryos as the model, not only identifies FGF signaling as the main target for correcting IVF-associated epigenetic errors, but also highlights the importance of oviductal paracrine factors in supporting early embryonic development and improving in vitro culture system.
... In recent years, the number of children conceived after IVF has increased, and it would appear that most of them are healthy (Servick, 2014). Nonetheless, recent evidence has raised a slight concern about pregnancy complications (Racowsky, 2002), perinatal mortality, preterm birth (Bergh, Ericson, Hillensjö, Nygren, & Wennerholm, 1999), lower birth weight and defects (Reefhuis et al., 2008), and imprinting disorders (Batcheller, Cardozo, Maguire, DeCherney, & Segars, 2011) in IVF pregnancies. In addition, it has been reported that IVF children may have an increased incidence of childhood and adulthood complications such as epilepsy, congenital malformations, diabetes, hypothyroidism, asthma, and hypertension (Batcheller et al., 2011;S. ...
Background:
In vitro fertilization (IVF) is a well-accepted procedure which has been utilized for the treatment of infertile patients. As embryos at early stages of development are very vulnerable, the IVF conditions may influence genetic and epigenetic regulation of preimplantation mouse embryo.
Methods:
We assessed the effect of IVF on the expression of developmental and implantation related miRNAs (miR-21, miR-93, miR-24, and let-7a), their common presumptive target (Stat3), and miRNA biogenesis pathway genes (Drosha, Dgcr8, Exportin-5, Dicer, and Ago2). in vivo 8-cell and blastocysts were compared to IVF embryos. Expression levels of miRNAs, Stat3, and miRNA biogenesis pathway genes were evaluated by qRT-PCR in in vivo (n = 8) and IVF (n = 4) embryos.
Results:
The expression levels of let-7a and Stat3 were significantly reduced in IVF blastocyst when compared with in vivo (p = .004 and p = .009, respectively). Nevertheless, the IVF procedure did not influence the expression levels of miRNA biogenesis pathway components in 8-cell and blastocyst embryos.
Conclusions:
Downregulation of let-7a and developmental related transcription factor, Stat3, in IVF mouse blastocysts may affect preimplantation development and implantation of embryos. Moreover, the genes of the miRNA biogenesis pathway were not changed in preimplantation mouse embryos through the IVF procedure.
... Implantation involves a complex sequence of events that are crucial to pregnancy success. However, the implantation process efficiency is relatively low (Gnoth, Godehardt, Godehardt, Frank-Herrmann, & Freundl, 2003;Racowsky, 2002). Indeed, despite the marked improvements in IVF success rates over the last few decades, there is still a wide range of patients, in whom high-quality embryos are transferred, that experiences RIF, which may be caused by factors related to the endometrium (Timeva, Shterev, & Kyurkchiev, 2014). ...
For the present study we asked whether the endometrial fluid lipidomic may be a useful approach to predict endometrial receptivity in freeze‐all cycles. For this case‐control study, endometrial fluid samples were collected from 41 patients undergoing freeze‐all cycles. Samples were split depending on the pregnancy outcome: Positive‐Group (n=24) and Negative‐Group (n=17). Data were acquired by the MALDI‐TOF mass spectrometry. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS‐DA) were applied. A list of potential biomarker ion ratios was obtained and the values were used to build a ROC curve to predict pregnancy success. The lipid categories were attributed by Lipidmaps database. Ion‐ratios were established according to their correlations and used for the analysis. The PCA showed a tendency of separation between the studied groups, while the PLS‐DA was able to clearly distinguish them. Fifteen ratios (13 hyper‐represented in the Negative and two hyper‐represented in the Positive‐Group) were selected according to their importance for model prediction. These ratios were used to build the ROC curve, which presented an AUC of 84.0% (95%CI: 69.2–97.4%, p=0.009). These findings suggest that lipidomic profiling of endometrial fluid may be a valuable tool for identifying the time interval comprising the window of implantation.
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... The most significant aberration leading to embryonic loss is the first meiotic division resulting in non-disjunction and aneuploidy. Autosomal trisomies, including trisomies 13, 15, 16, 18 and 21, account for the most common spontaneous pregnancy losses [17]. In addition, there is a significant absence of autosomal monosomies in spontaneous human abortions, suggesting that these aberrations must be lethal to embryonic viability very early post-fertilization, in fact before the first missed menstrual period. ...
Objectives:
Multiple pregnancies are known to be associated with adverse maternal and perinatal complications. How-ever, data regarding the outcomes of spontaneously reduced twin pregnancies are limited. In the current study we aimed to evaluate the consequences of the vanishing twin syndrome (VTS) in dichorionic diamniotic twin pregnancies for both mother and baby in our perinatal center.
Material and methods:
A total of 711 pregnancies were included into the study. 51 cases of vanishing twin syndrome constituted Group 1, 235 cases of normal twins constituted Group 2, and 425 singleton pregnancies formed Group 3. The pregnancies that had multifetal reduction and monochorionic twinning were excluded from both study group and twin control group. The collected data were as follows: age, gravidity, parity, gestational week at birth, delivery route, birth weight, obstetric complications, and maternal and perinatal outcomes.
Results:
No significant difference was observed between the groups regarding mean maternal age (p > 0.05). Mean birth weight, gestational age at birth and preterm birth ratio were significantly lower in the Group 2 when compared with Group 1 and Group 3 (all p < 0.001). Adverse perinatal outcomes including very low birth weight (VLBV) and low Apgar scores were more common in Group 1 (p < 0.05), but no significant difference was found between the groups in terms of neona-tal intensive care unit admission and perinatal mortality ratios (p > 0.05). Obstetric complications such as preeclampsia, gestational diabetes and intrauterine growth restriction were significantly higher in Group 2 than in Group 1 and Group 3 (all p < 0.05). However, severe maternal morbidities were similar among three groups (p = 0.141).
Conclusions:
VTS is seems to be associated with VLBV and low Apgar scores. However, the incidence of severe maternal and perinatal morbidity and mortality in pregnancies with VTS is similar to other pregnancies.
... It is widely used to treat human infertility, and is also used for animal breeding and propagation. While IVF is generally considered a safe and successful technology, there are several IVF-associated safety concerns, such as pregnancy loss, preterm birth, lower birthweight and birth defects, and higher risk of age-related disorders, such as heart disease, diabetes, or hypertension (Bergh et al., 1999;Racowsky, 2002;Schieve et al., 2002;Klemetti et al., 2006;Ceelen et al., 2008;Reefhuis et al., 2009;Wen et al., 2012;Hansen et al., 2013;Servick, 2014). ...
Study hypothesis: How does in vitro fertilization (IVF) alter promoter DNA methylation patterns and its subsequent effects on gene expression profiles during placentation in mice?
Study finding: IVF-induced alterations in promoter DNA methylation might have functional consequences in a number of biological processes and functions during IVF placentation, including actin cytoskeleton organization, hematopoiesis, vasculogenesis, energy metabolism and nutrient transport.
What is known already: During postimplantation embryonic development, both embryonic and extraembryonic tissues undergo de novo DNA methylation, thereby establishing a global DNA methylation pattern, and influencing gene expression profiles. Embryonic and placental tissues of IVF conceptuses can have aberrant morphology and functions, resulting in adverse pregnancy outcomes such as pregnancy loss, low birth weight, and long-term health effects. To date, the IVF-induced global profiling of DNA methylation alterations, and their functional consequences on aberrant gene expression profiles in IVF placentas have not been systematically studied.
Study design, samples/materials, methods: Institute for Cancer Research mice (6 week-old females and 89 week-old males) were used to generate in vivo fertilization (IVO) and IVF blastocysts. After either IVO and development (IVO group as control) or in vitro fertilization and culture (IVF group), blastocysts were collected and transferred to pseudopregnant recipient mice. Extraembryonic (ectoplacental cone and extraembryonic ectoderm) and placental tissues from both groups were sampled at embryonic day (E) 7.5 (IVO, n = 822; IVF, n = 795) and E10.5 (IVO, n = 324; IVF, n = 278), respectively. The collected extraembryonic (E7.5) and placental tissues (E10.5) were then used for high-throughput RNA sequencing (RNA-seq) and methylated DNA immunoprecipitation sequencing (MeDIP-seq). The main dysfunctions indicated by bioinformatic analyses were further validated using molecular detection, and morphometric and phenotypic analyses.
Main results and the role of chance: Dynamic functional profiling of high-throughput data, together with molecular detection, and morphometric and phenotypic analyses, showed that differentially expressed genes dysregulated by DNA methylation were functionally involved in: 1) actin cytoskeleton disorganization in IVF extraembryonic tissues, which may impair allantois or chorion formation, and chorioallantoic fusion; 2) disturbed hematopoiesis and vasculogenesis, which may lead to abnormal placenta labyrinth formation and thereby impairing nutrition transport in IVF placentas; 3) dysregulated energy and amino acid metabolism, which may cause placental dysfunctions, leading to delayed embryonic development or even lethality; 4) disrupted genetic information processing, which can further influence gene transcriptional and translational processes.
Limitations, reasons for caution: Findings in mouse placental tissues may not be fully representative of human placentas. Further studies are necessary to confirm these findings and determine their clinical significance.
Wider implications of the findings: Our study is the first to provide the genome-wide analysis of gene expression dysregulation caused by DNA methylation during IVF placentation. Systematic understanding of the molecular mechanisms implicated in IVF placentation can be useful for the improvement of existing assisted conception systems to prevent these IVF-associated safety concerns.
Study funding and competing interest(s): This work was supported by grants from the National Natural Science Foundation of China (No. 31472092), and the National High-Tech R&D Program (Nos. 2011AA100303, 2013AA102506). There was no conflict of interest.
... However, there are associated small, but worrying, safety concerns. These include embryonic loss (Racowsky, 2002), preterm birth and perinatal mortality (Bergh et al., 1999), lower birthweight and birth defects (Schieve et al., 2002;Wen et al., 2012;Hansen et al., 2013), and increased risk of childhood and adulthood diseases (Klemetti et al., 2006;Ceelen et al., 2008;Reefhuis et al., 2009). While these adverse outcomes are thought to be due to the IVF manipulation and culture process, the precise mechanisms remain largely unclear. ...
Study question:
Does in vitro fertilization (IVF) induce comprehensive and consistent changes in gene expression associated with mitochondrial biogenesis and function in mouse embryos from the pre- to post-implantation stage?
Summary answer:
IVF-induced consistent mitochondrial dysfunction in early mouse embryos by altering the expression of a number of mitochondria-related genes.
What is known already:
Although IVF is generally safe and successful for the treatment of human infertility, there is increasing evidence that those conceived by IVF suffer increased health risks. The mitochondrion is a multifunctional organelle that plays a crucial role in early development. We hypothesized that mitochondrial dysfunction is associated with increased IVF-induced embryonic defects and risks in offspring.
Study design, size, duration:
After either IVF and development (IVO groups as control) or IVF and culture (IVF groups), blastocysts were collected and transferred to pseudo-pregnant recipient mice. Both IVO and IVF embryos were sampled at E3.5, E7.5 and E10.5, and the expression profiles of mitochondria-related genes from the pre- to post-implantation stage were compared.
Participants/materials, setting, methods:
ICR mice (5- to 6-week-old males and 8- to 9-week-old females) were used to generate IVO and IVF blastocysts. Embryo day (E) 3.5 blastocysts were transferred to pseudo-pregnant recipient mice. Both IVO and IVF embryos were sampled at E3.5, E7.5 and E10.5 for generating transcriptome data. Mitochondria-related genes were filtered for dynamic functional profiling. Mitochondrial dysfunctions indicated by bioinformatic analysis were further validated using cytological and molecular detection, morphometric and phenotypic analysis and integrated analysis with other high-throughput data.
Main results and the role of chance:
A total of 806, 795 and 753 mitochondria-related genes were significantly (P < 0.05) dysregulated in IVF embryos at E3.5, E7.5 and E10.5, respectively. Dynamic functional profiling, together with cytological and molecular investigations, indicated that IVF-induced mitochondrial dysfunctions mainly included: (i) inhibited mitochondrial biogenesis and impaired maintenance of DNA methylation of mitochondria-related genes during the post-implantation stage; (ii) dysregulated glutathione/glutathione peroxidase (GSH/Gpx) system and increased mitochondria-mediated apoptosis; (iii) disturbed mitochondrial β-oxidation, oxidative phosphorylation and amino acid metabolism; and (iv) disrupted mitochondrial transmembrane transport and membrane organization. We also demonstrated that some mitochondrial dysfunctions in IVF embryos, including impaired mitochondrial biogenesis, dysregulated GSH homeostasis and reactive oxygen species-induced apoptosis, can be rescued by treatment with melatonin, a mitochondria-targeted antioxidant, during in vitro culture.
Limitations, reasons for caution:
Findings in mouse embryos and fetuses may not be fully transferable to humans. Further studies are needed to confirm these findings and to determine their clinical significance better.
Wider implications of the findings:
The present study provides a new insight in understanding the mechanism of IVF-induced aberrations during embryonic development and the increased health risks in the offspring. In addition, we highlighted the possibility of improving existing IVF systems by modulating mitochondrial functions.
Study funding/competing interests:
This work was supported by grants from the National Natural Science Foundation of China (No. 31472092), and the National High-Tech R&D Program (Nos. 2011|AA100303, 2013AA102506). There was no conflict of interest.
... We believe a reason for the lack of significant imprinting errors in this region despite all the environmental and parental risks is possibly because of the lower rates of implantation in ART. Many of the embryos introduced to the uterus during ART fail to implant (Racowsky, 2002) and we believe that this failure to implant may be buffering any serious imprinting abnormalities; any affected embryo would fail to develop further due to an inability to implant. Regardless of whether or not the implantation in ART can act as a quality control step, there is still a potential for imprinting defects in this group. ...
... It is estimated that approximately 1.5 million ART cycles are performed annually worldwide, resulting in 350 000 live births [1,2]. ART can lead to a series of health problems, including embryonic loss [3], preterm birth, and perinatal mortality [4], low birth weight [5], pre-eclampsia (PE) [6], and an increased risk of childhood and adulthood diseases [7,8]. However, the mechanisms that underlie these problems are still unclear. ...
As the interface between the mother and the developing fetus, the placenta is believed to play an important role in assisted reproductive technology (ART)-induced aberrant intrauterine and postnatal development. However, the mechanisms underlying aberrant placentation remain unclear, especially during the extra-embryonic tissue development and early stage of placental formation. Using a mouse model, this investigation provides the first comparative proteomic analysis of in vivo (IVO) and in vitro produced (IVP) extra-embryonic tissues and placentas after in vivo fertilization and development, or in vitro fertilization and culture, respectively. We identified 165 and 178 differentially expressed proteins (DEPs) between IVO and IVP extra-embryonic tissues and placentas on Embryonic Day 7.5 (E7.5) and E10.5, respectively. Many DEPs were functionally associated with genetic information processing, such as impaired de novo DNA methylation, as well as post-transcriptional, translational and post-translational dysregulation. These novel findings were further confirmed by global hypomethylation, and a lower level of correlation was found between the transcriptome and proteome in the IVP groups. In addition, numerous DEPs were involved in energy and amino acid metabolism, cytoskeleton organization and transport, and vasculogenesis and angiogenesis. These disturbed processes and pathways are likely to be associated with embryonic intrauterine growth restriction, an enlarged placenta and impaired labyrinth morphogenesis. This study provides a direct and comprehensive reference for the further exploration of the placental mechanisms that underlie ART-induced developmental aberrations.
... Pregnancy loss is the most common complication of human gestation, occurring in as many as 75% of all women trying to have children. Roughly onehalf of conceptions in humans result in pregnancy loss, with losses occurring most frequently in the first two weeks of gestation [14]. The failure to establish pregnancy in humans and animals is due to both embryonic and maternal factors [13,15,16]. ...
Infertility and subfertility are important and pervasive reproductive problems in both domestic animals and humans. The majority of embryonic loss occurs during the first three weeks of pregnancy in cattle and women due, in part, to inadequate endometrial receptivity for support of embryo implantation. To identify heifers of contrasting fertility, serial rounds of artificial insemination (AI) were conducted in 201 synchronized crossbred beef heifers. The heifers were then fertility classified based on number of pregnancies detected on day 35 in four AI opportunities. Heifers, classified as having high fertility, subfertility or infertility, were selected for further study. The fertility-classified heifers were superovulated and flushed, and the recovered embryos were graded and then transferred to synchronized recipients. Quantity of embryos recovered per flush, embryo quality, and subsequent recipient pregnancy rates did not differ by fertility classification. Two in vivo-produced bovine embryos (stage 4 or 5, grade 1 or 2) were then transferred into each heifer on day 7 post-estrus. Pregnancy rates were greater in high fertility than lower fertility heifers when heifers were used as embryo recipients. The reproductive tracts of the classified heifers were obtained on day 14 of the estrous cycle. No obvious morphological differences in reproductive tract structures and histology of the uterus were observed in the heifers. Microarray analysis revealed differences in the endometrial transcriptome based on fertility classification. A genome-wide association study, based on SNP genotyping, detected 7 moderate associations with fertility across 6 different chromosomes. Collectively, these studies support the idea that innate differences in uterine function underlie fertility and early pregnancy loss in ruminants. Cattle with defined early pregnancy success or loss is useful to elucidate the complex biological and genetic mechanisms governing endometrial receptivity and uterine competency for pregnancy.
... The cause of fragmentation in human embryos is unknown. Embryos with severe and persistent fragmentation are less likely to be viable, and severe fragments observed at the one-to two-cell stage are fatal [75], whereas small amounts of fragmentation do not correlate with negative outcomes [76]. The common practice of assigning grades to embryos is based largely on blastomere fragmentation, and poor embryo grades are strongly associated with lowered implantation and pregnancy rates. ...
Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of MII oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO2 in air for 2.25 h. Sperm were then assessed for motility, viability and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the 8-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in 2-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to 4- and 8-cell stage was significantly lower for embryos generated with ROS- treated sperm than for controls. All embryos produced from ROS -treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of 2-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the 2-cell stage of development.
... It is debatable whether the genomic, physiological and behavioral effects would still occur for gestations with a regular density of embryos in the female genital tract, which is important for the timing of parturition in a polytocous species like the mouse (McLaren, 1970 ). In humans, the first and foremost aim of fertility clinics is to ensure the delivery of healthy babies, and only in suborder, to increase the pregnancy rates—a sensible ranking of priorities since the natural fecundity of our species is low anyway (Racowsky, 2002). However, when losses are high during early development, it seems unrealistic that the surviving embryos have remained completely unaffected. ...
Study question:
Do different human ART culture protocols prepare embryos differently for post-implantation development?
Summary answer:
The type of ART culture protocol results in distinct cellular and molecular phenotypes in vitro at the blastocyst stage as well as subsequently during in vivo development.
What is known already:
It has been reported that ART culture medium affects human development as measured by gestation rates and birthweights. However, due to individual variation across ART patients, it is not possible as yet to pinpoint a cause-effect relationship between choice of culture medium and developmental outcome.
Study design, size, duration:
In a prospective study, 13 human ART culture protocols were compared two at a time against in vivo and in vitro controls. Superovulated mouse oocytes were fertilized in vivo using outbred and inbred mating schemes. Zygotes were cultured in medium or in the oviduct and scored for developmental parameters 96 h later. Blastocysts were either analyzed or transferred into fosters to measure implantation rates and fetal development. In total, 5735 fertilized mouse oocytes, 1732 blastocysts, 605 fetuses and 178 newborns were examined during the course of the study (December 2010-December 2011).
Participants/materials, setting, methods:
Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and recipients for embryo transfer, respectively. In vivo fertilized B6C3F1 oocytes were allowed to cleave in 13 human ART culture protocols compared with mouse oviduct and optimized mouse medium (KSOM(aa)). Cell lineage composition of resultant blastocysts was analyzed by immunostaining and confocal microscopy (trophectoderm, Cdx2; primitive ectoderm, Nanog; primitive endoderm, Sox17), global gene expression by microarray analysis, and rates of development to midgestation and to term.
Main results and the role of chance:
Mouse zygotes show profound variation in blastocyst (49.9-91.9%) and fetal (15.7-62.0%) development rates across the 13 ART culture protocols tested (R(2)= 0.337). Two opposite protocols, human tubal fluid/multiblast (high fetal rate) and ISM1/ISM2 (low fetal rate), were analyzed in depth using outbred and inbred fertilization schemes. Resultant blastocysts show imbalances of cell lineage composition; culture medium-specific deviation of gene expression (38 genes, ≥ 4-fold) compared with the in vivo pattern; and produce different litter sizes (P ≤ 0.0076) after transfer into fosters. Confounding effects of subfertility, life style and genetic heterogeneity are reduced to a minimum in the mouse model compared with ART patients.
Limitations, reasons for caution:
This is an animal model study. Mouse embryo responses to human ART media are not transferable 1-to-1 to human development due to structural and physiologic differences between oocytes of the two species.
Wider implications of the findings:
Our data promote awareness that human ART culture media affect embryo development. Effects reported here in the mouse may apply also in human, because no ART medium presently available on the market has been optimized for human embryo development. The mouse embryo assay (MEA), which requires ART media to support at least 80% blastocyst formation, is in need of reform and should be extended to include post-implantation development.
... Despite improvements in human embryo culture conditions and implantation rates, there still remains an inherent degree of embryonic loss during in vitro culture (Patrizio and Sakkas, 2009). As a result, disparities in embryo quality are routinely observed within individual embryo cohorts and poor-quality embryos (PQEs) are a common and accepted by-product of most IVF/ICSI cycles (Racowsky, 2002). ...
BACKGROUND Human embryonic stem cells (hESCs) are most commonly derived from the inner cell mass (ICM) of blastocyst stage embryos.
While the majority of hESC lines originate from good-quality embryos donated after cryogenic storage, poor-quality embryos
(PQEs) not suitable for clinical use have also been shown to generate hESC. This provides a newfound function for embryos
that would otherwise be discarded following IVF or ICSI. Owing to their lack of clinical importance, however, data on the
poorest embryos in a cohort go largely unreported in the literature. It is therefore of interest to better understand the
availability of PQEs from IVF/ICSI cycles and to determine their ability to develop into blastocysts with good-quality ICMs
for use in hESC derivation. In this study, we investigate the influence of patient parameters and embryo cohort on PQE incidence,
blastocyst development, ICM quality and successful hESC derivation from donated PQEs.
... In particular, in the absence of sufficient knowledge of human preimplantation embryo development, success rates of IVF have remained relatively low; even though apparently healthy-looking embryos are selected for embryo transfer (see Glossary, Box 1) back to the mother, they frequently fail to implant (see Box 3). To improve the odds of pregnancy given the uncertainty regarding embryo developmental potential, multiple embryos are transferred in some clinics, resulting in multiple births and the associated complications of low-birth weight, prematurity and, in some cases, the need to reduce fetal number for the health of the mother or siblings (Racowsky, 2002). Generally, the best IVF clinics attempt to optimize the pregnancy rate while minimizing adverse outcomes. ...
Understanding human pre-implantation development has important implications for assisted reproductive technology (ART) and for human embryonic stem cell (hESC)-based therapies. Owing to limited resources, the cellular and molecular mechanisms governing this early stage of human development are poorly understood. Nonetheless, recent advances in non-invasive imaging techniques and molecular and genomic technologies have helped to increase our understanding of this fascinating stage of human development. Here, we summarize what is currently known about human pre-implantation embryo development and highlight how further studies of human pre-implantation embryos can be used to improve ART and to fully harness the potential of hESCs for therapeutic goals.
... One of the main objectives for ART, which is a presently used policy in several European IVF clinics, is to reduce the number of multiple gestations by single embryo transfer. Current morphological-and growth-related criteria that are commonly used to assess embryo viability on Day 3 may both underestimate or overestimate embryo potential (Racowsky, 2002). Given the uncertainties associated with evaluation at Day 3, some clinics have turned to extended culture regimens to improve the assessment of embryo implantation potential (Milki et al., 2000;Gardner et al., 2004). ...
Time-lapse observation presents an opportunity for optimizing embryo selection based on morphological grading as well as providing novel kinetic parameters, which may further improve accurate selection of viable embryos. The objective of this retrospective study was to identify the morphokinetic parameters specific to embryos that were capable of implanting. In order to compare a large number of embryos, with minimal variation in culture conditions, we have used an automatic embryo monitoring system.
Using a tri-gas IVF incubator with a built-in camera designed to automatically acquire images at defined time points, we have simultaneously monitored up to 72 individual embryos without removing the embryos from the controlled environment. Images were acquired every 15 min in five different focal planes for at least 64 h for each embryo. We have monitored the development of transferred embryos from 285 couples undergoing their first ICSI cycle. The total number of transferred embryos was 522, of which 247 either failed to implant or fully implanted, with full implantation meaning that all transferred embryos in a treatment implanted.
A detailed retrospective analysis of cleavage times, blastomere size and multinucleation was made for the 247 transferred embryos with either failed or full implantation. We found that several parameters were significantly correlated with subsequent implantation (e.g. time of first and subsequent cleavages as well as the time between cleavages). The most predictive parameters were: (i) time of division to 5 cells, t5 (48.8-56.6 h after ICSI); (ii) time between division to 3 cells and subsequent division to 4 cells, s2 (≤ 0.76 h) and (iii) duration of cell cycle two, i.e. time between division to 2 cells and division to 3 cells, cc2 (≤ 11.9 h). We also observed aberrant behavior such as multinucleation at the 4 cell stage, uneven blastomere size at the 2 cell stage and abrupt cell division to three or more cells, which appeared to largely preclude implantation.
The image acquisition and time-lapse analysis system makes it possible to determine exact timing of embryo cleavages in a clinical setting. We propose a multivariable model based on our findings to classify embryos according to their probability of implantation. The efficacy of this classification will be evaluated in a prospective randomized study that ultimately will determine if implantation rates can be improved by time-lapse analysis.
... Our lack of knowledge of human embryo development carries a heavy burden in terms of health and finance [1]. Consider that the success rate of assisted reproductive techniques such as IVF is just 25% and that ca. ...
Although some aspects of human embryo development are conserved with those of other species, including the mouse, many aspects such as the timing of reprogramming and occurrence in the absence of transcription, duration of transcriptional silence and identity of genes with modulated expression in the oocyte to embryo transition, appear to be unique. Yet, frequently, the only data available for understanding the programs of early embryo development is that derived from model or agricultural species. We suggest that a specific understanding of basic aspects of human embryo development can affect a two-fold positive impact: 1) We can improve the health of a substantial subset of patients who seek assisted reproduction by improving diagnostics of viable embryo development in the clinic and, 2) we can use the information we gather to improve derivation and diagnosis of pluripotent stem cell lines (including reference or gold-standard human embryonic stem cell (hESC) lines and closely-related induced pluripotent stem cell (iPSC) lines) and their fates in novel basic and clinical applications.
... Third and finally, given that embryo developmental potential can be assessed with a combination of cytokinetic and mitotic parameters in the first two cleavage divisions, it may be feasible to translate these basic studies to clinical applications. Current morphological and growth criteria that are commonly used to assess embryo viability on day 3 in assisted reproduction clinics may both underestimate and overestimate embryo potential, with well-documented consequences, such as multiple births, the need for fetal reduction and miscarriage 34 . Given the uncertainties associated with evaluation at day 3, some clinics have turned to longer culture to assess embryo potential, as embryos transferred at the blastocyst stage have a higher implantation rate compared with embryos transferred at day 3 (refs. ...
We report studies of preimplantation human embryo development that correlate time-lapse image analysis and gene expression profiling. By examining a large set of zygotes from in vitro fertilization (IVF), we find that success in progression to the blastocyst stage can be predicted with >93% sensitivity and specificity by measuring three dynamic, noninvasive imaging parameters by day 2 after fertilization, before embryonic genome activation (EGA). These parameters can be reliably monitored by automated image analysis, confirming that successful development follows a set of carefully orchestrated and predictable events. Moreover, we show that imaging phenotypes reflect molecular programs of the embryo and of individual blastomeres. Single-cell gene expression analysis reveals that blastomeres develop cell autonomously, with some cells advancing to EGA and others arresting. These studies indicate that success and failure in human embryo development is largely determined before EGA. Our methods and algorithms may provide an approach for early diagnosis of embryo potential in assisted reproduction.
... In fact, only a small proportion of the oocyte population can develop to healthy embryos after fertilization and healthy fetuses after transfer. Although multiple factors are at play in determining pregnancy outcome in assisted reproduction including age, sperm quality (male factor), fertilization capacity and number of embryos transferred, the effect of fertilization rate appears to be of less significance [15] and that intrinsic deficiencies of the oocyte and/or embryo account for greater than 50% of failed conceptions [16] . These findings suggest that the developmental competence of the oocytes is a major determinant in the establishment of successful pregnancy in assisted reproduction. ...
Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte.
Immature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC) of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC). Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array.
The result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox) and nerve growth factor receptor associated protein 1 (Ngfrap1), which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2), which is involved in the regulation of extracellular matrix organization and biogenesis.
The data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and the developmental competence of oocytes. This finding suggests that the most differentially expressed gene, lysyl oxidase, may be a candidate biomarker of oocyte health and useful for the selection of good quality oocytes for assisted reproduction.
... In the laboratory, the improvements have been primarily at the level of media, allowing development of embryos in vitro and selection at a more advanced stage (Bavister, 1969; Goddard and Pratt, 1983; Chatot et al., 1989; Leese, 1989; 1990; 1991; 1995; Lawitts and Biggers, 1993; Leese et al., 1993; Quinn et al., 1995; Gardner and Lane, 1997). However, the reports of high clinical pregnancy rates can come at an extreme cost to the patient and society in that these are achieved by the use of multiple embryos in transfer, leading to a world-wide escalating incidence of both twin and high-order multiple pregnancies (Racowsky, 2002). The practice of replacing multiple embryos arises from the fact that the reported implantation rates for human embryos range from as low as 8% to a maximum of approximately 40%, implying that between 60 and 92% of all embryos transferred do not implant. ...
There is a need for more accurate embryo selection in human assisted reproduction, if the goal of reducing the number of embryos used in embryo transfer is to be realized. Furthermore, any selection strategy should be non-invasive if the embryos are to be used in embryo transfer. Currently, the strategy is selection by one to three parameters in the cleaving- and blastocyst-stage embryo, sometimes with additional pronuclear selection. It is clear that no one system is ideal, as the vast majority of transferred embryos do not implant. As the health of the embryo is largely dictated by the originating gametes, the very early events in oocyte development should be considered. This review will point to the early biological events in the unfertilized and fertilized oocyte that can be scored non-invasively and which can have a profound effect on the later developmental stages. Using a sequential scoring system, with emphasis on the oocyte, a system for selecting the most viable single embryo for transfer may hopefully be achieved.
... The most signi®cant intrinsic factor contributing to embryonic loss is aberrations in the ®rst meiotic division resulting in non-disjunction and aneuploidy. Trisomies 13, 15, 16, 18 and 21 account for the most common autosomal trisomies in spontaneous pregnancy losses (Racowsky, 2002). It may be that in the future preimplantation genetic screening of embryos prior to transfer may reduce early pregnancy wastage resulting from aneuploidy (Handyside et al., 1999; Munne  et al., 1999). ...
The risk of spontaneous first trimester abortion is estimated to be between 10 and 20%. Although it is common knowledge that the incidence of abortion decreases as pregnancy progresses, exact data in relation to the duration of pregnancy are scarce.
We reviewed 1597 clinical IVF/ICSI pregnancies with known outcome and tabulated the number of miscarriages or fetal demise per intervals of 2 weeks. We furthermore compared the outcome in terms of fetal survival of 1200 singleton pregnancies with that of 397 twin pregnancies.
The overall incidence of non-ongoing singleton pregnancies was 21.7%. Fetal death, after positive heart activity had been recorded, occurred in 12.2% of singleton pregnancies. The overall incidence of spontaneous abortion in twin pregnancies was 17.1% (12.1% vanishing twins and 5.0% complete miscarriages). The incidence of miscarriage in the twin pregnancies, expressed per gestational sac, was 11.1%. Once fetal heart activity was present, the risk of abortion (per gestational sac) was 7.3%, which is significantly lower than that in singleton pregnancies.
Our data give an estimate of the probability of miscarriage or fetal demise at any given period of the first trimester both for singleton and twin pregnancies. Twin pregnancies after IVF have a better potential for survival than singleton pregnancies.
The establishment of repositories of frozen semen, for the conservation of agricultural genetic resources, is not a simple matter of collecting and freezing semen in the hope that one day it will be suitable for use in an artificial insemination procedure. Important genetic issues need to be considered; for example, how many samples should be stored and from how many individuals? Aside from these, many biological and logistic issues must be considered. Cryopreservation technology does not work equally well in all species, often because of anatomical differences in the female reproductive tract leading to significant variability in the number of spermatozoa needed in order to achieve an acceptable conception rate. Moreover, spermatozoa from different species are not equally susceptible to cryoinjury. However, it is also emerging that semen samples from individuals within a species are also of different quality; several studies have revealed that these differences reflect the quality of DNA within the spermatozoon itself and also the efficacy of biochemical functions, including metabolic and signalling systems, within individual cells. As new possibilities to select spermatozoa for insemination arise, especially the use of flowsorting for gender selection, these issues may become more significant. In this article we interpret the way in which some of this new information may impact upon the practical implementation of genetic resource conservation.
Recurrent Pregnancy Loss (RPL) affects 2–5% of the population, and 10% of all the patients undergoing IVF treatment end up with Recurrent Implantation Failure. Nearly 50% of these patients are not aware of the cause of their pregnancy loss and so their cases are often referred to as “unexplained.” This chapter discusses the definitions, incidence, types, and classifications of RPL and RIF. It also talks about the importance of biochemical pregnancy and the concept of RPL attributable to chance.KeywordsRecurrent pregnancy lossRPLRecurrent implantation failureRIFUnexplained infertilityDefinition of RPLDefinition of RIFIncidence of RPL/RIFTypes of RPLBiochemical pregnancyRPL by chance
Problem
Human infertility affects 15–20% of reproductive-age couples and it is mitigated by assisted reproductive technology (ART) approaches. Poor biological viability of embryos contributes to implantation failure and live birth rate (LBR). This study is aimed to examine whether or not embryo-secreted soluble human leukocyte antigen-G (sHLA-G) is (i) associated with developing embryos and (ii) able to predict successful pregnancy outcome.
Method of study
A retrospective, multicentric study using 539 human embryo spent medium samples (E-SMs), analysed for sHLA-G levels by ELISA. Correlation analysis was performed on sHLA-G levels with developing embryonic stages, their quality scores and pregnancy outcome in terms of LBR.
Results
Of 539 E-SMs analysed, 445 had detectable sHLA-G (83%) with levels varying within and across clinics and, between stages of embryonic development. Levels of sHLA-G (ng/mL) were significantly (P < .05) different in E-SMs of cleavage-stage embryos versus blastocysts. There was an insignificant correlation between the sHLA-G levels and morphology scores of embryos. But, sHLA-G levels showed a positive correlation with grades of blastocysts and importantly, its levels were significantly (P < .05) higher in live-birth vis-a-vis no-birth cases. Also, levels were higher in live-births out of blastocysts-ETs versus cleavage-stage-embryo transfers. Altered levels were observed with embryos, which resulted in miscarriages. Overall, a significant (P < .0001) association of sHLA-G with live births was observed.
Conclusion
Embryo-derived sHLA-G can be a valuable embryo viability, independent, biomarker, which can predict live-birth outcome and it could be useful as an adjunct to existing criteria for elective single embryo transfer.
Gastrulation is a phase in early mammalian development when the three germ layers are generated and body plan is formed. Although well-studied in mice, much less is known about gastrulation in humans. Due to the lack of access to primary human tissue for study and experimental manipulation, as well as legal and ethical constraints surrounding the use of human embryos, a dissection of the molecular and cellular mechanisms that underlie this process in humans has proven elusive. Non-human primates (NHPs), owing to their relatedness to human species, comprise a tantalizing alternative model system for understanding human biology. Two recent studies have established novel systems to study monkey embryos for 20 days, demonstrating landmark events of early primate embryogenesis with possible relevance to human development. Most strikingly, cells grown in the dish closely resembled cells in in vivo embryos, suggesting that embryo development in a dish might actually be equivalent to that which occurs in vivo. In this piece, the author discusses the tremendous potential of these new methods to unveil insights into mechanisms that mediate primate embryo development. Moreover, repurposing the extended monkey embryo culture methods to create human-monkey embryonic chimeras would aid the development of strategies to create human organs inside livestock species. Finally, the ethical and regulatory issues that emerge from reconsideration of extending time limits for human embryo culture beyond 14 days or primitive streak formation are also briefly considered.
There are few treatments for patients with recurrent pregnancy loss (RPL) or recurrent implantation failure (RIF). Women with RPL and unexplained infertility have lower T regulatory cell (T reg ) expression when compared to fertile controls. A murine model has been developed with depletion of regulatory T cells (DEREG) after administration of diphtheria toxin (DT), resulting in smaller litter sizes, secondary to embryo implantation failure. Numerous murine studies have shown that adoptive transfer of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T regs from donors improves litter sizes in DEREG mice with depleted T regs . Our hypothesis is that DEREG mice treated with a single dose of DT will deplete T regs and subsequently decrease litter sizes and that treatment with rapamycin (sirolimus; Pfizer) during the time of embryo implantation will increase T regs and restore litter sizes nearly back to normal levels. Syngeneic mating of DEREG mice after depletion of T regs resulted in smaller litter sizes and this defect was reversed when these DEREG mice were treated with rapamycin at the time of embryo implantation. The importance of T regs at the time of embryo implantation has been well established and immunotherapy treatments, such as rapamycin (mammalian target of rapamycin inhibitor), may prove to be an effective treatment for patients with RPL, RIF, or unexplained infertility with low T reg .
Non-human primates (NHPs) have been shown to be highly relevant models for studies of human infectious disease and fundamental pathophysiology. NHPs have also been used to address issues related to women's health, assisted reproduction, fetal well-being, and miscarriage, and have been shown to be highly relevant models for studies on assisted reproduction, gamete biology, endocrine control of gamete production, and behavior. This chapter outlines the importance of NHP models for development of understanding of human development and infertility. We discuss the advantages of the models for applications that include assisted reproductive technology (ART), as well as for studies into early embryo development. We compare developmental landmarks of timing of embryo cleavage and cytokinesis in primates and discuss some research applications for understanding these processes.
The implantation rate of embryos in human assisted reproduction (ART) is low with between 10 and 40% of transferred embryos implanting. It has been estimated that the overall natural fecundity in normal healthy couples is approximately 20–25% in any one cycle, not too different from that seen in ART, however the expectation of doing better than nature has crept into the practice of ART (Edwards and Beard, 1997). To increase success rates, some practitioners have resorted to multiple embryo replacements with a resulting escalation of high-order pregnancies worldwide without a corresponding increase in the overall implantation rate (Racowsky, 2002). Some countries have mandated limits to the number of embryos that can be replaced (Ludwig et al., 2000) and others have proposed guidelines and recommendations to reduce the risk of higher order gestations. To comply with the regulations and limit the number of embryos replaced while not reducing the probability of implantation requires reliable methods of selecting embryos with the most potential for implantation. If it is assumed that human embryos obtained by in vitro fertilization (IVF) at different ART centers have after similar ranges of competence after fertilization, a 4-fold difference in implantation rate is troublesome and suggests that clinic-specific practices and procedures have an important impact on outcome. This chapter reviews the relevant findings pertaining to how signs of high competence can be recognized at the earliest stages of human embryo development in vitro and describes criteria that, if adopted by IVF programs, should significantly improve outcome in centers where ongoing pregnancy rates persist at low levels.
Since the first pregnancy after in vitro fertilization and embryo transfer (IVF-ET) over a million pregnancies have been achieved worldwide by IVF and its modifications. On a per-cycle basis, the results of IVF are similar to the fecundability of natural conception cycles in the general population. The cumulative success rate is higher when IVF is attempted on a repetitive basis. Pregnancy rates have consistently improved over time. By 2002, the delivery rate per retrieval was 32%. However, the incidence of twins and high order multiple pregnancies (ie. triplets or more) has risen over the past two decades. From 1980 to 1997, the annual number of live born babies from twin gestations rose 52%, while the number of high order gestations increased 404%. This rise was mainly due to increased use of fertility drugs for ovulation induction, superovulation, and IVF. In the USA, 1997 data showed that approximately 18% of high order gestations were spontaneous, 38% due to ovulation induction procedures, and 43% attributable to IVF. Compared to natural ovulation and conception, IVF increases the chance of having twins 20-fold and triplets/quadruplets 400%. Often, infertility patients consider the birth of twins acceptable, or even desirable, since it results in an family increase after years of infertility. A number of studies have reported an increase in the adverse perinatal outcome of pregnancies obtained with IVF-ET. Multiple gestations are at increased risk of fetal, neonatal, and maternal complications, as well as complete pregnancy loss, when compared to singleton pregnancies. Neonatal complications are primarily the result of preterm delivery. Several strategies attempting to control the frequency of high order multiples have been implemented. The overriding aim of these strategies is to transfer fewer embryos of higher quality to maximize the pregnancy rate and to minimize the risk of high order multiple gestation. National legislation in several European countries has proven to be more effective in reducing the high order pregnancy rate than practice guidelines in the USA. Maternal age is important because women under age 35 achieved excellent birth and multiple gestation rates with trensfer of two embryos, while women over 35 years of age required transfer of three embryos to achieve similar results. The ability to select only the highest quality embryos would allow transfer of fewer of them achieving acceptable birth rates. Day 5 transfer (blastocyst stage) of only one or two blastocysts need to be transferred to maintain overall success rates while decreasing the high order multiple pregnancy rate. Utilization of single embryo transfer will likely increase as method of predicting embryo viability and to reduce high order pregnancy mounts. Strategies to reduce the high order multiple gestation rate start from ovulation induction including close monitoring of follicle development, a strict cycle cancellation policy and aspiration of supernumerary follicles. After multifetal pregnancy reduction, pregnancies with three or less fetuses have fewer pregnancy losses, increased gestational age, and higher weight. Selective termination has been established as a safe and effective means for termination of one or two abnormal fetuses.
Background: During the past 25 years SBTE has organized and held annual meetings. Such events have been proven essential to demonstrate how important Brazilian scientific activities are in the context of reproductive biotechnologies. The consolidation of a wide range of distinct research groups, usually supported by graduate programs mostly at federal universities, has allowed the qualification of a large number of M.Sc. and Ph.D. professionals in the field of animal reproduction. The demand by the Brazilian agribusiness, supported by the new frontiers in cattle production, such as the regions of the Midwest and the vast periphery of the Amazon basin, where currently more than 60% of the national cattle herd is maintained, allowed the successful use of reproductive biotechnologies to spread superior genetics to herds, mainly focused on Zebu breeds. Review: The use of reproductive biotechnologies in developed countries during the 50s and 60s of last century quickly attracted the interest of Brazilian reproductive physiologists, who were interested in the cryopreservation of semen and in the artificial insemination of domestic animals. During the 70s, important advancement attained in procedures involving the physiology of the female made possible the use of bovine embryo transfer technology in a large-scale. Then, in 1974, the organization known as International Embryo Transfer Society (IETS) was founded in the U.S.A. The IETS emerged as one of the main global forum for discussions on reproductive biotechnologies. The annual meetings of the IETS enabled the participation of a keen group of young Brazilian veterinarians and animal reproduction specialists who were interested in knowing and applying new knowledge on that specific area. The support of private sponsors, associated with a new scenario in graduate academic training, funded mostly by CAPES and CNPq, enabled the training of Brazilian colleagues in universities and research centers in the U.S.A. and countries in Western Europe, in particular in Germany and France. The use of embryo transfer in Brazil, funded by private enterprise, has achieved success in 1979 with the birth of the first calf in the State of São Paulo, in the city of Sorocaba. The calf was named EUREKA by our colleague Jorge Nicolau. In the 80s, the participation of Brazilian colleagues in the annual events of the IETS in the U.S.A. increased significantly. In July 1985, during the Brazilian Congress on Animal Reproduction, in Belo Horizonte, a first meeting of the recent created organization known as Brazilian Embryo Transfer Society (SBTE) was held by its pioneering members, with the election of the Society's first board at that time. Conclusion: This paper present a historical and scientific perspective of activities undertaken by SBTE, mainly through the themes and discussions carried out in their annual meetings.
Significance
The human sex ratio has long interested cell biologists, developmental biologists, demographers, epidemiologists, evolutionary biologists, gynecologists, and statisticians. Nonetheless, the trajectory of the human sex ratio from conception to birth has been poorly characterized. We present the most comprehensive analysis of this trajectory ever done. Our dataset is the largest ever assembled to estimate the sex ratio at conception and is the first, to our knowledge, to include data from 3- to- 6-d-old embryos, induced abortions, chorionic villus sampling, amniocentesis, and fetal deaths and live births. Our results indicate that the sex ratio at conception is unbiased, the proportion of males increases during the first trimester, and total female mortality during pregnancy exceeds total male mortality; these are fundamental insights into early human development.
Detecting intrauterine exposure to environmental pollutants, based on an increased number of malformations, has only been
successful with a few teratogens. Nor does the number of spontaneous abortions represent a more reliable indicator, since
a precise record of early abortions is not available. A greater vulnerability to prenatal damage leading to abortion is evident
in male embryos/fetuses than in female. The newborn sex ratio (birth rate of boys/girls) is a very stable parameter in healthy
populations. Its decrease has been reported after exposure to some harmful environmental factors. We document a decrease in
the male birth fraction in the Czech Republic after the Chernobyl disaster in 1986. The absolute numbers of male and female
births were determined in each of 600 consecutive months from 1950 to 1999. There were always more newborn boys than girls,
except in November 1986, when the number of male births significantly decreased. This deficit in male births might have resulted
from the spontaneous abortion of male embryos/fetuses during weeks 8-12 of pregnancy, as a consequence of their increased
exposure to radiation, in particular to the radionuclide iodine131. We propose using the newborn sex ratio as a further tool
for the standard evaluation of reproductive quality. Combined analyses of the incidence of newborn malformations, spontaneous
abortions and stillbirths, intrauterine growth retardation and the newborn sex ratio will help to compensate for the imperfections
associated with each of these parameters individually and will provide a more complete understanding of the extent of prenatal
risk induced by environmental factors.
Posthumous reproduction refers to a deliberate decision to produce a child after one or both potential biological parents
die (1). For purposes of this chapter, we are also using this term to refer to reproduction when one or both of the biological parents
is in a permanent unconscious condition (2). Posthumous reproduction may involve several distinct levels of physician involvement: (1) in the procurement (“harvesting”)
of the biological specimens; (2) in the preparation and/or storage of those specimens; and (3) in the use of the specimens
for reproduction. Posthumous reproduction may include the use of “banked” specimens that were knowingly procured and stored
by explicit consent of the individual. However, this chapter focuses on a more recent phenomenon of harvesting and use of
specimens collected after death or a diagnosis of a permanent unconscious condition of the “donating” individual (3). The medical and legal communities have debated what physicians should or should not do in response to requests for posthumous
reproduction for a number of years without achieving a complete consensus. In the United States, there are currently no uniform
laws or regulations for governing assisted reproduction (4). Through a series of questions listed below, this chapter poses the common questions and examines the trends in posthumous
reproduction.
Teratology is the branch of medical science which studies the contribution of the environment to abnormal prenatal growth as well as morphological or functional developmental defects. Despite some pioneering research, the clinical and scientific interest in clinical teratology only developed because of the rubella pandemics in 1941 and the thalidomide tragedy in the late 1950s. The following decades have seen a definition of criteria for proof of human teratogenicity, the classification of drugs used in pregnancy, and the development of the “Teratogen Information Services” in developed countries.
Teratogens are chemical, physical or infectious agents, or a maternal status/disease whose prenatal exposure during the pregnancy can provoke developmental defects. Susceptibility to teratogenic agents depends on the combination of several factors, including the genotype of the mother and/or of the fetus, dosage, the gestational period at exposure, pharmacokinetics and the pharmacodynamic of the substance.
The methodology for the identification of teratogens includes animal studies, prospective epidemiologic studies and retrospective epidemiologic studies. The criteria for “proof” of human teratogenicity have not been defined although some criteria have been proposed and applied in the clinical/animal/epidemiologic studies. At present, some international classifications of drugs used in pregnancy are
Successful establishment and maintenance of pregnancy can be attained only through optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. In particular, very little information exists for the posthatching embryo and elongating conceptus. This period of development is arguably the most important, as approximately 40% of all embryonic loss occurs between Days 8 and 17 of pregnancy in cattle. Here, we describe the global transcriptome profile of the bovine conceptus at five key stages of its pre- and peri-implantation growth (Days 7, 10, 13, 16, and 19) using state-of-the-art RNA sequencing techniques. More than 287 million reads were generated at the five stages, and more than 22 700 unique transcripts were detected. Analysis of variance followed by self-organizing maps identified differentially regulated (P < 0.05) genes organized in nine gene clusters forming a sequential transcript dynamics across these developmental stages. Of particular interest, genes in clusters 3 (n = 236) and 6 (n = 1409) were significantly up-regulated on Days 16 and 19, suggesting a role in maternal recognition and initiation of implantation. This transcriptome analysis of the bovine conceptus will provide a blueprint of the dynamic changes in gene expression occurring during maternal recognition and implantation and will complement existing knowledge of the temporal changes in the endometrial transcriptome, thus facilitating a better understanding of conceptus-maternal cross talk during the peri-implantation period of pregnancy.
The controversies surrounding embryonic stem cell research have prompted scientists to invent beyond restrictive national policy and moral concerns. The impetus behind these reports comes from different sources, including individually held moral beliefs, societal pressures and resource constraints, both biological and financial. Along with other contributions to public policy such as advocacy or public testimony, experimentation and scientific curiosity are perhaps more natural responses scientists use to surmount impediments to research. In a research context, we review the history of the first stem cell discoveries, and describe scientific efforts leading up to recent reports of pluripotent lines made without the use of human embryos and eggs. We argue that despite the promise of these new lines, we must not lose sight of fundamental questions remaining at the frontiers of embryology and early human development. The answers to these questions will impact studies of genetics, cell biology and diseases such as cancer, autoimmunity and disorders of development. Human embryonic stem cell research is barely a decade old. The recent pace of discovery--in spite of federal restrictions--is testament to the potential of these cells to uncover some of biology's most intractable mysteries.
Murine 2-cells embryos were isolated from murine oviducts at laboratory and transferred into Ham's F-10 medium containing 0.1 mg mL(-1) streptomycin and 100 IU mL(-1) penicillin G and supplemented with 3 mg mL(-1) bovine serum albumin (BSA) or different concentrations of bovine follicular fluid (bFF) and estrous cow serum (ECS). Significantly higher (p<0.05) > or =4-cell embryos were developed when embryos were cultured 20% bFF (84.33%) comparing to 10 and 15% bFF (48.33 and 69.33%) as well as 3 mg mL(-1) BSA (65.66%). Morula rates were also lower in 10% bFF (22.33%) comparing to the other groups and were similar in 15 and 20% bFF (62.66 and 72.33% morula rates) as well as BSA containing media (55.33%). The highest (p<0.05) blastocyst rates were obtained in medium containing 20% bFF (64.33%) and the lowest belonged to 10% bFF (15%) comparing to 15% bFF (33.66%) or 3 mg mL(-1) BSA. When embryos were cultured in ECS, no significant different was observed in different culture media (76.66, 72.33, 82.5 and 65.66% > or =4-cell embryos in 10, 15 and 20% bFF and 3 mg mL(-1) BSA, respectively). Morula and blastocyst rates were also similar in all groups (32.33, 41.66 and 66.25 and 55.33% morula rates and 15.33, 27, 44.50 and 29.66% blastocyst rates for 10, 15 and 20% bFF and 3 mg mL(-1) BSA, respectively). The results of the present study demonstrated that 20% bFF could be substituted for BSA when in vitro culture of murine embryos is carried.
The developmental competence of embryos cloned from somatic cells depends on the cellular event and molecular process, such as separation of chromosomes and reorganization of spindle after nuclear transfer. Centrosome, the main microtubule organizing centers in a cell, is crucial for reorganization of spindle and normal separation of chromosomes during mitosis. Aberrant of centrosomes will lead to aneuploidy of blastomere and developmental failure of embryo. This paper expounded the situation of animal somatic cell nuclear transfer (SCNT) and biological functions of centrosome and analyzed the inheritance mechanism of centrosome during gametogenesis and fertilization. Additionally, the study condition of centrosome and its associated proteins in SCNT embryos were introduced, which provided a new clue to study the de-velopmental abnormality of cloned embryos and animals.
In 1999, the approximately 90,000 in vitro fertilization procedures accounted for 98% of all types of assisted reproductive technologies in the United States. Since 1992, when Congress recognized a public health interest in reporting accurate and timely information about pregnancy success rates for infertility treatments, success has been defined as a live birth after an assisted reproductive technology cycle, regardless of the number of live-born infants per delivery. Because of pressures to achieve success, often more than one pre-embryo is transferred per cycle, frequently resulting in multifetal pregnancy reduction or multiple births. Twin and higher order births associated with assisted reproductive technology have increased significantly since 1980. Although births resulting from assisted reproductive technology amount to less than 1% of all live births, they now account for about a third of all twin births and more than 40% of triplets and higher number births in the United States. Although multiple births fit the current definition of success, they create much higher risks for maternal and infant morbidity and mortality, contributing to more than $640 million in excess initial hospital costs during the year 2000 alone. With recent improvements in assisted reproductive technology procedures that increase the likelihood of delivery after the transfer of just one pre-embryo per cycle, it is time to re-examine how success is measured. Assisted reproductive technology success should be redefined to be the proportion of cycles resulting in a singleton, live birth.
Many strategies have been proposed for the selection of viable embryos for transfer in human assisted reproduction. These have included morphological scoring criteria for day 1, 2, 3 and 5 embryos or combinations of these. Other strategies have used predictors such as timing of certain key events, as with early cleavage to the 2-cell, development to the 8-cell stage or patterns of fragmentation. All have shown some correlations with implantation. However, the overall success of these methods is still limited, with over 50% of all transferred embryos failing to implant. The use of pronuclear oocyte morphology has shown correlations with implantation and development to the blastocyst stage. The key aspects of pronuclear scoring, namely the presence of a cytoplasmic halo, the orientation of the nuclei in relation to the polar bodies and the size, number and pattern of distribution of nucleolar precursor bodies (NPB) in the nuclei were related to day 2,3 and 5 development, rate of development and day 3 and 5 morphology in a retrospective study. The pattern of the NPB or Z-score and the presence/absence of a halo had a significant effect on the rate of development on day 3 and day 5 and on the overall embryo morphology score. Low Z-score resulted in slow development, poor blastocyst formation and low morphology scores. The absence of a halo also resulted in slow and poor development, poor morphology, increased fragmentation and increased numbers of poor Z-scored embryos. The use of PN scoring can help predict embryos that have poor developmental potential, aid in early selection and may indicate the health of the oocyte.
Extended embryo culture together with amelioration of embryo selection methods and embryo culture conditions have allowed a substantial increase on both pregnancy and implantation rates. However, uterine embryo transfers are still performed after 2 to 6 days of egg retrieval. In this paper, we show the results of two studies, one prospective study comparing IVF outcome of day 2 and day 3 embryo transfers, and a retrospective study looking at blastocyst transfers versus day 3 embryo transfers in our egg donation program. Also, we test the predictive value of the presence of three or more seven cell-stage embryos on day 3 of development on blastocyst formation and pregnancy rates.
In-vitro fertilization (IVF) embryos are selected for transfer on the basis of morphology and rate of development. However, when a number of embryos have similar characteristics, the selection of the best embryos is left to chance. Recently, we proposed a simple, novel method to overcome this problem, based on pre-selection of embryos cleaving early to the two-cell stage. In this study we have adopted the same method to choose embryos fertilized after intracytoplasmic sperm injection (ICSI). Fertilized embryos that had cleaved to the two-cell stage by 27 h post-injection were designated as 'early cleavage' embryos, while those that had not yet reached the two-cell stage were designated as 'no early cleavage'. In all cases, the early cleavage embryos were transferred when available. Early cleavage was observed in 54 (61.4%) of the 88 cycles assessed. There were significantly (P = 0.04) more clinical pregnancies in the early cleavage group, 14/54 (25.9%), compared with the no early cleavage group 2/34 (3.2%). No differences between the groups were found when comparing key parameters (age, stimulation protocol and semen characteristics) of the couples. Using the ICSI technique, we have shown that early cleavage to the two-cell stage is not influenced by the timing of fertilization, and is more likely due to intrinsic factors within the oocyte or embryo that promote embryo cleavage after fertilization.
This retrospective study of 1001 in-vitro fertilization (IVF) cycles included a consecutive series of single transfers (n = 341), dual transfers (n = 410) and triple transfers (n = 250) where all the transferred embryos in each cycle were of identical quality score and identical cleavage stage. In our 2 day culture system, transfer of 4-cell embryos resulted in a significantly higher implantation rate and pregnancy rate (23 and 49%) compared with 2-cell embryos (12 and 22%) and 3-cell embryos (7 and 15%). Furthermore, the transfer of 4-cell embryos resulted in a significantly higher pregnancy rate compared with embryos that had cleaved beyond the 4-cell stage (28%). The implantation rate (21%) and pregnancy rate (43%) after transfer of embryos of score 1.0 were significantly higher than after transfer of embryos of score 2.0 (14 and 32% respectively). Transferring embryos of score 2.1 resulted in significantly higher implantation rates (26%) and similar pregnancy rates compared with score 1.0. Transferring embryos of score 2.2-3.0 resulted in a significantly lower implantation rate (5%) and pregnancy rate (15%). A striking finding was that embryos of quality score 2.0 had a significantly lower implantation rate compared with embryos of quality score 1.0 and 2.1 and a significantly lower pregnancy rate compared to embryos of quality score 1.0. We also found a lower implantation rate and pregnancy rate when transferring 3-cell embryos. These findings may indicate periods of increased sensitivity to damage during the cell cycle. In conclusion, these results substantiate the idea of the superiority of 4-cell embryos and demonstrate that minor amounts of fragments in the embryo may not be of any importance. These findings may call for a shift when weighing the two main morphological components (quality score and cleavage stage) in the sense that reaching a 4-cell cleavage stage even with the presence of a minor amount of fragments should be preferred to a 2-cell embryo with no fragments.
A number of non-invasive methods have been proposed to evaluate embryo viability in human in-vitro fertilization programmes.
In addition to biochemical analyses, a common method for the selection of embryos prior to transfer involves assessment of
embryo quality and morphology. We propose a new method to evaluate embryo viability based on the timing of the first cell
division. Fertilized embryos that had cleaved to the 2-cell stage 25 h post-insemination were designated as 'early cleavage'
embryos while the others that had not yet reached the 2-cell stage were designated as 'no early cleavage'. In all cases the
early cleavage embryos were transferred when available. Early cleavage was observed in 27 (18.9%) of the 143 cycles assessed.
There were significantly (chi2 = 4.0; P = 0.04) more clinical pregnancies in the early cleavage group, 9/27 (33.3%), compared
with the no early cleavage group, 17/116 (14.7%). No difference was found when comparing key parameters (age, stimulation
protocol and semen characteristics) of couples belonging to both groups, pointing to an intrinsic property or factor(s) within
the early cleaving embryos. We propose 'early cleavage' as a simple and effective non-invasive method for selection and evaluation
of embryos prior to transfer.
In human in-vitro fertilization (IVF) embryos are routinely transferred to the uterus on day 2 or day 3 of development. Resultant implantation and pregnancy rates are disappointingly low, with only approximately 10% of embryos transferred leading to a live birth. The ability to culture embryos to the blastocyst stage should help to resolve this problem by synchronizing the embryo with the female reproductive tract, and by identifying those embryos with little developmental potential. Co-culture has offered a possible means of producing blastocysts capable of high implantation rates. However, recent developments in the field of embryo physiology and metabolism have led to the formulation of new sequential serum-free culture media capable of supporting the development of viable blastocysts in several mammalian species, including the human. It is therefore proposed that blastocyst transfer should be considered for routine use in human IVF. The high viability of blastocysts cultured in the appropriate sequential media means that fewer embryos are required for transfer to achieve a pregnancy, culminating in fewer multiple births. Furthermore, the development of suitable non-invasive tests of embryo viability should further increase the overall success of human IVF by the ability to select before transfer those blastocysts most able to establish a pregnancy.
In human in-vitro fertilization (IVF), embryos are routinely transferred to the uterus on either day 2 or day 3 of development, resulting in a 10-15% implantation rate. However, in other mammalian species, the transfer of cleavage stage embryos, which normally reside in the oviduct, to the uterus results in a significantly lower implantation rate compared with blastocysts. It is therefore proposed that, in order to increase implantation rates in human IVF, one has to move to extended culture and transfer at the blastocyst stage. The transfer of blastocysts will not only help synchronize the embryo with the female tract but will facilitate the identification of those embryos with little or no developmental potential. In order to culture viable blastocysts it is important to use more than one culture medium to cater for the changing requirements of the preimplantation embryo as it develops and differentiates. If sequential culture media are not used, one can obtain blastocysts but their resultant viability is low. The use of sequential serum-free media in human IVF has resulted in > 50% of embryos becoming blastocysts with an implantation rate of approximately 50%. Further advances in human embryo culture should come from the replacement of protein with the glycosaminoglycan hyaluronate, which is more suitable than albumin in supporting implantation in the mouse, and which will eliminate biological variation and possible contamination from blood products. With the routine culture of human blastocysts will come the introduction of non-invasive tests of embryo viability, capable of identifying those blastocysts most likely to develop from a given cohort. As the implantation rate of blastocysts is higher than that of the cleavage stage embryo, fewer embryos will be required for transfer in order to establish a successful pregnancy, thereby reducing the number of multiple gestations and increasing the overall efficiency of human IVF.
Testing shows that most laboratories conducting human gamete and embryo culture have air quality and sources of contamination that exceed the levels measured in homes, businesses and schools. The sources of these contaminants have been shown to be either from activities outside the laboratory, or emitted from materials used in the facility, such as compressed gas, cleaning and sterilizing agents, plastic and stored materials. Both the laboratory structure and the air handling systems may affect the air composition. The significance of these findings is being validated by the accumulation of field case studies and now by assay procedures. Products given off by road sealant were shown to have accumulated in one of the examined laboratories, adjacent to a large re-surfaced parking area. Aldehydes such as acrolein, hexanal, decanal, pentanal and others were detected at elevated concentrations that were statistically significant. Since it is not appropriate to add potentially suspect chemicals to human embryos, we used a mouse-model to study the effect of acrolein. The growth of mouse embryos was significantly affected after acrolein was added at different concentrations to the culture environment. The physiological effect was noted at concentrations in the low ppm range. The testing end-point of embryo death must still be considered to be a crude basis for evaluating toxicological effects, since it involves addition of compounds to culture media and unprotected growth until the blastocyst stage. The findings may, however, support observations of decreased pregnancy rate following exposure of human embryos to aldehydes or other adverse conditions. With proper engineering and material selection, it is possible to reduce such contamination. The usefulness of this approach for controlling aldehydes has been demonstrated by decreasing levels in the laboratory to below those of the outside air.
This retrospective study was undertaken to determine whether further developmental progression of twopronucleated (2PN) zygotes
can be predicted by a single, non-invasive examination of pronuclei, with the use of criteria based on the number and distribution
of nucleolar precursor bodies in each pronucleus. The normal range of pronuclear variability was defined by analysis of zygotes
giving rise to embryos transferred in 100%-implantation cycles (pattern 0). Morphological patterns differing from pattern
0 were classified as patterns 1–5. The frequency of developmental arrest of pattern 0 zygotes was only 8.5% as compared with
31.6, 21.9, 30.0, 20.5 and 24.1% for patterns 1–5 respectively. Relationships of pronuclear patterns with blastomere multinucleation
and cleaving embryo morphology were also noted. Clinical pregnancy was achieved in 22 of 44 (50%) treatment cycles in which
at least one pattern 0 embryo was transferred, but only in two of 23 (9%) cycles in which only pattern 1–5 embryos were transferred.
These data present new evaluation criteria which can be used to predict the developmental fate of human embryos as early as
the pronuclear stage, without requiring repeated observations or an exact timing of pronuclear zygote inspection. Further
prospective study is needed for clinical validation of these criteria.
Chromosomal abnormalities are responsible for a great deal of embryo wastage, which is reflected, at least partially, in decreased
implantation and increased miscarriage in older women. To address this problem the transfer of only chromosomally normal embryos
previously selected by preimplantation genetic diagnosis (PGD) has been proposed. We designed a multi-centre in-vitro fertilization
(IVF) study to compare controls and a test group that underwent embryo biopsy and PGD for aneuploidy. Patients were matched
retrospectively, but blindly, for average maternal age, number of previous IVF cycles, duration of stimulation, oestradiol
concentrations on day +1, and average mature follicles. All these parameters were similar in test and control groups. Only
embryos classified as normal for those chromosomes were transferred after PGD. The results showed that the rates of fetal
heart beat (FHB)/embryo transferred between the control and test groups were similar. However, spontaneous abortions, measured
as FHB aborted/FHB detected, decreased after PGD (P < 0.05), and ongoing pregnancies and delivered babies increased (P < 0.05) in the PGD group of patients. Two conclusions were obtained: (i) PGD of aneuploidy reduced embryo loss after implantation;
(ii) implantation rates were not significantly improved, but the proportion of ongoing and delivered babies was increased.
Three hypotheses were tested: (i) the distance between first and second polar bodies (PB) may relate to embryo morphology,
(ii) that the orientation of pronuclei (PN) relative to PB may relate to embryo morphology, (iii) that the placement of a
spermatozoon in a fixed plane relative to the first PB [intracytoplasmic sperm injection (ICSI)] may alter PN/PB orientation
relative to in-vitro fertilization (IVF). A total of 251 two pronuclear (2PN) embryos (124 ICSI, 127 IVF) from 64 patients
was studied. Angles were measured between the PN axis and the nearest PB (α), the furthest PB (β), and between the two PB
(γ). On day 2, the morphological grades of embryos were recorded. γ ranged from 0 to 150° and was not significantly different
for ICSI or IVF embryos of different grades; however, an unusual distribution of γ suggested different populations of oocytes.
The first hypothesis was rejected. α and β ranged from 0 to 90°: α did not relate significantly to embryo grade, but β increased
significantly with decreasing quality of ICSI embryos (P < 0.05) and the total group (P < 0.01), supporting hypothesis (ii). The difference in β between ICSI and IVF embryos was not significant, so hypothesis
(iii) was unproven. Significant differences between ICSI and IVF embryos in PN positions, irregular cleavage, and cleavage
failure were noted.
The association between oocyte morphology and subsequent fertilization rate and embryo quality in intracytoplasmic sperm injection
(ICSI) is subject to considerable controversy. This retrospective study was carried out to investigate a possible prognostic
value of first polar body morphology with regard to fertilization rate and embryo quality. A total of 70 consecutive ICSI
cases was included in this study. The results showed that classification based on first polar body morphology revealed a significant
correlation with fertilization rate (P < 0.025) and embryo quality (P < 0.001). Cytoplasmic criteria showed no correlation in this respect. Present data indicate that ICSI of oocytes with intact
well-shaped first polar bodies yields higher fertilization rates and higher quality embryos.
Preimplantation genetic diagnosis (PGD) and subsequent embryo development was evaluated in 72 couples presenting at our centre
for intracytoplasmic sperm injection (ICSI) due to severe male factor. The embryo biopsies were performed in Ca2+/Mg2+-free medium. These patients were further divided into those with advanced maternal age (AMA, n = 49) and those with recurrent implantation failure (RIF, n = 23). Fluorescence in-situ hybridization (FISH) was carried out on 329 blastomeres (91.3%) with probes for the X, Y, 13,
18 and 21 chromosomes. The chromosomal abnormality rate was 41.3% with no significant difference between the AMA and RIF groups.
Aneuploidy accounted for the majority (72.8%) of chromosomal abnormalities. Out of 329 embryos, 84.2% had cleaved after 24
h and 15.1% had arrested. Embryos were transferred in 70 patients and 22 pregnancies were achieved (31.4% with an ongoing
pregnancy rate of 28.5%). There were no significant differences between the pregnancy rates of the AMA and RIF groups (32.5
and 30% respectively). Therefore PGD should be offered to patients with AMA and RIF. Furthermore, the use of Ca2+/Mg2+-free medium during the blastomere biopsy facilitates the procedure, while further embryo cleavage, ongoing pregnancies and
healthy births are possible.
In 126 selected patients treated with clomiphene and human menopausal gonadotrophin, 154 embryos were transferred into the uterus 41–50 h after in-vitro insemination. Fifty per cent of embryos implanted. The rate of cleavage was inversely related to the success of embryo transfer; implantation failures occurred for 69 and 46% of embryos with more than or less than four cells, respectively {p <0.05). Cleavage rate was not related to the viscosity of the cumulus cell mass, serum supplementation of culture medium, nor to the age or genetic factors of the woman. There was a trend towards smaller follicles, and a significant (p <0.05) tendency for patients developing numerous follicles to yield faster-cleaving embryos. These relationships were not found in 50 other embryos obtained from patients treated with pure FSH.
Objective To examine the effect of pituitary suppression and the women’s age on embryo viability and uterine receptivity. Design Retrospective analysis of 394 embryo transfers (ET) after in vitro fertilization (IVF). Setting Community hospital IVF program from 1986 to 1990. Patients Three groups were studied: women Interventions Pituitary suppression was achieved in groups 1 and 3 with daily leuprolide acetate starting in the luteal phase; human menopausal gonadotropin and progesterone were given intramuscularly. Main Outcome Measures Ongoing and multiple ongoing pregnancy rates (PRs) were compared in the three groups. A mathematical model of implantation was used to estimate embryo viability and uterine receptivity. Results Ongoing and multiple ongoing PRs per ET in group 1 (28.6% and 12.3%) were significantly higher than the corresponding rates in group 2 (16.9% and 2.4%) and in group 3 (16.9% and 3.4%). Implantation analysis revealed higher embryo viability without change in uterine receptivity with pituitary suppression (group 1 versus 2). Decrease in both embryo viability and uterine receptivity was noted in women > 40 (group 1 versus 3). Conclusions (1) Pituitary suppression improved implantation outcome by increasing embryo viability with no change in uterine receptivity and was associated with a high multiple PR in women 40 both embryo viability and, to a lesser extent, uterine receptivity were decreased; (3) transfer of a larger number of embryos in older patients may improve IVF outcome without excessive risk of multiple pregnancy.
Between 1980 and 1997 the number and rate of twin, triplet, and higher-order multiple births has climbed at an unprecedented pace within the United States. The number of live births in twin deliveries rose 52% between 1980 and 1997, and the number of live births in triplet and higher-order multiple pregnancies increased an astounding 404%. In sharp contract, single births during the same time period rose 6%. The last several years has witnessed an even more remarkable aspect to the trend in multiple pregnancies in that there was nearly a 1,000% increase in the incidence of multiple gestation among women 45–49 years of age between 1980 and 1997 (1). These extraordinary increases in the incidence of multiple pregnancy is a public health concern due to the considerable medical, social, and financial consequences of multiple gestation.
Emerging data from animal and human experimentation underscore the uniqueness and dynamic nature of both the physiology of the human preimplantation embryo and of the environment in which early development occurs. The incorporation of these principles into the design of contemporary culture strategies now allows for the efficient production in vitro of viable human embryos of advanced developmental stages, through the use of sequential culture systems without the need for whole serum or somatic cell support.
The frequency with which congenital malformations occur varies considerably from country to country and, within countries, from area to area and social class to social class. For example, neural-tube malformations are five times commoner in England and Wales than in Japanl and nearly three times commoner in the coalmining valleys of South Wales than along its coastal plain2: in Scotland they are three times commoner in infants born into social class v than in infants born into social classes I and II.1 From these and similar observations it has been inferred that environmental teratogens must be at work, but in making such an inference few research workers stop to consider what relation, if any, the prevalence of malformations at birth bears to their incidence at the time they are laid down-early in the first trimester of pregnancy.
Objective:
To investigate the effect of improved air quality on IVF and subsequent embryo development.
Design:
Retrospective cohort study.
Setting:
Hospital-based IVF facility composed of an anteroom, a cleanroom, and an adjacent operating room.
Patient(s):
Two-hundred seventy-five couples requesting IVF between 1993 and 1997.
Intervention(s):
None.
Main outcome measure(s):
Particle counts (sizes 0.3, 0.5, 1.0, and 5.0 microm); IVF rates; and embryo quality (stage and grade).
Result(s):
Clinical pregnancy rates decreased from 35% in 1993 to 16% in 1994 (numerous construction odors were detected during 1994) and increased steadily after the cleanroom was built (rates for 1995-1997 were 20%, 32%, and 59%, respectively). Fertilization rates decreased between 1993 (74%) and 1994 (60%) and then steadily increased after cleanroom installation (62% in 1995, 71% in 1996, and 69% in 1997). The proportion of embryos past the four-cell stage decreased from 66% in 1993 to 61% in 1994 but then increased steadily in the years after the cleanroom was built (78%, 77%, and 83% in 1995, 1996, and 1997, respectively). During the same 5-year period, there were no differences in embryo quality or number of embryos transferred.
Conclusion(s):
Construction of a Class 100 cleanroom improved air quality and IVF rate and increased the number of embryos past the four-cell stage available for transfer.
In order to achieve a clinical pregnancy rate higher than that achieved following initial adoption of in-vitro fertilization embryo transfers, more than one embryo is transferred. This has led to a substantial increase in unwanted multiple pregnancy rates with IVF as compared with natural conception. What is therefore required is a simple, clinically useful embryo scoring system, to reflect embryo developmental potential, which will enable the selection of the optimal number of embryos to transfer in order to achieve the maximum pregnancy rate with a low incidence of high order multiple pregnancies. We believe that the Cumulative Embryo Score (CES) achieves these aims. On the day of embryo transfer the grade of each embryo transferred was multiplied by the number of blastomeres to produce a score for each embryo, and summation of the scores obtained for all the embryos transferred gave the CES. The grouped pregnancy rates obtained rose as the CES increased to maximum of 42. A continued increase in the CES above 42 did not result in any further rise in the pregnancy rate. However, an analysis of all our IVF pregnancies showed that the multiple pregnancy rate continued to rise above a CES of 42. By restricting the CES per embryo transfer to 42, 78% of triplet pregnancies and 100% of the quadruplet IVF pregnancies could have been predicted and potentially avoided.
To examine the effect of pituitary suppression and the women's age on embryo viability and uterine receptivity.
Retrospective analysis of 394 embryo transfers (ET) after in vitro fertilization (IVF).
Community hospital IVF program from 1986 to 1990.
Three groups were studied: women less than 40 years with pituitary suppression (group 1) and without pituitary suppression (group 2); women 40 years of age and older with pituitary suppression (group 3).
Pituitary suppression was achieved in groups 1 and 3 with daily leuprolide acetate starting in the luteal phase; human menopausal gonadotropin and progesterone were given intramuscularly.
Ongoing and multiple ongoing pregnancy rates (PRs) were compared in the three groups. A mathematical model of implantation was used to estimate embryo viability and uterine receptivity.
Ongoing and multiple ongoing PRs per ET in group 1 (28.6% and 12.3%) were significantly higher than the corresponding rates in group 2 (16.9% and 2.4%) and in group 3 (16.9% and 3.4%). Implantation analysis revealed higher embryo viability without change in uterine receptivity with pituitary suppression (group 1 versus 2). Decrease in both embryo viability and uterine receptivity was noted in women greater than 40 (group 1 versus 3).
(1) Pituitary suppression improved implantation outcome by increasing embryo viability with no change in uterine receptivity and was associated with a high multiple PR in women less than 40; (2) in women greater than 40 both embryo viability and, to a lesser extent, uterine receptivity were decreased; (3) transfer of a larger number of embryos in older patients may improve IVF outcome without excessive risk of multiple pregnancy.
To determine if a simple morphological classification of embryos was predictive of subsequent pregnancy.
Prospective case series.
University-based in vitro fertilization (IVF) program.
Consecutive embryo transfer (ET) cycles (n = 206).
Embryos were classified into three grades: (1) equal-size blastomeres with no fragmentation; (2) unequal-size blastomeres; and (3) evidence of fragmentation.
Embryo quality, age, indication for IVF, and stimulation protocol were evaluated for their effect on pregnancy rates (PR's).
In cycles in which the best embryo transferred was grade 3, 2, or 1, the clinical PRs per ET were 0% (0/11 cycles), 12.8% (6/47 cycles, P less than 0.05), and 21.8% (32/148 cycles, P less than 0.05), respectively. When one, two, or three or more grade 1 embryos were replaced, the clinical PRs per ET were 15.6%, 16.3%, and 40% (P less than 0.05), respectively. Using logistic regression, embryo quality (P = 0.0011) and patient's age (P = 0.0044) were the only variables that affected PRs.
The transfer of more than two good quality embryos had a positive effect, patient's age had a negative effect on PRs after IVF-ET.
An important factor influencing the pregnancy rate after in vitro fertilization-embryo transfer (IVF-ET) appears to be the number of embryos transferred to the uterus. In this study, the influence of oocyte maturity and embryo quality on pregnancy rate was assessed in patients undergoing IVF-ET. Ovarian hyperstimulation was performed by human menopausal gonadotropin (hMG [n = 29]), clomiphene citrate (CC)/hMG (n = 81), and hMG/follicle-stimulating hormone (FSH [n = 13]) protocols. Oocyte maturity was graded on a scale from 1 to 5 based on the morphology of the ooplasm, cumulus mass, corona radiata, and membrana granulosa cells. Embryos were graded according to the symmetry of the blastomeres and the presence or absence of fragmentation. Mature preovulatory oocytes yielded the highest fertilization rates. No differences were found among the protocols in terms of fertilization rate, embryo quality, or pregnancy rate. When all protocols were combined, patients who conceived had a significantly higher number of embryos transferred than those who did not conceive (3.6 +/- 0.1 [mean = SEM] versus 2.7 +/- 0.1). When embryo quality was compared, there was no difference in the number of "B" embryos transferred between patients who conceived and those who did not (1.2 +/- 0.2 versus 1.2 +/- 0.1), but the patients who conceived had significantly more "A" embryos transferred (1.6 +/- 0.3 versus 0.8 +/- 0.1). These data suggest that the treatment protocol did not determine embryo quality. Furthermore, the increase in pregnancy rates seen with an increase in embryos transferred is the result of the transfer of more "A" embryos.
In 126 selected patients treated with clomiphene and human menopausal gonadotrophin, 154 embryos were transferred into the uterus 41-50 h after in-vitro insemination. Fifty per cent of embryos implanted. The rate of cleavage was inversely related to the success of embryo transfer; implantation failures occurred for 69 and 46% of embryos with more than or less than four cells, respectively (p less than 0.05). Cleavage rate was not related to the viscosity of the cumulus cell mass, serum supplementation of culture medium, nor to the age or genetic factors of the woman. There was a trend towards smaller follicles, and a significant (p less than 0.05) tendency for patients developing numerous follicles to yield faster-cleaving embryos. These relationships were not found in 50 other embryos obtained from patients treated with pure FSH.
The earliest stages of development in most animals, including the few mammalian species that have been investigated, are regulated by maternally inherited information. Dependence on expression of the embryonic genome cannot be detected until the mid two-cell stage in the mouse, the four-cell stage in the pig (J. Osborn & C. Polge, personal communication), and the eight-cell stage in the sheep. Information about the timing of activation of the embryonic genome in the human is of relevance not only to the therapeutic practice of in vitro fertilization and embryo transfer (IVF), but more importantly for the successful development of techniques for the preimplantation diagnosis of certain inherited genetic diseases. We describe here changes in the pattern of polypeptides synthesized during the pre-implantation stages of human development, and demonstrate that some of the major qualitative changes which occur between the four- and eight-cell stages are dependent on transcription. In addition, it appears that cleavage is not sensitive to transcriptional inhibition until after the four-cell stage.
A semi-quantitative and non-invasive method for scoring embryos obtained after in-vitro fertilization (IVF) has been defined, aiming at selection of embryos before transfer and at prognostic evaluation of IVF trials. Grading of embryos observed on the inverted microscope was essentially based on the amount of anucleate fragments expelled during early cleavage and on developmental speed. Embryos endowed with a high score were more often associated with pregnancy and in particular with the occurrence of multiple pregnancy. No difference was observed between scores attributed to embryos related to ongoing, aborted or chemical pregnancies. Average embryonic scores corresponding to double and triple transfers differed significantly in failures as well as pregnancies. The better quality of embryos replaced in triple transfers was also apparent from the significantly higher implantation rate per embryo observed in this group. From our results, five criteria including clinical data and embryonic scores can be derived for defining a high risk of multiple pregnancy prior to transfer. It might be warranted to replace only two embryos when these conditions are fulfilled.
We studied the relationships between "pretransfer" parameters (number of follicles, oocytes, and embryos) and the result (occurrence and quality of pregnancy) obtained by transferring one, two, or three embryos. The analysis concerns 186 pregnancies compared with 186 implantation failures. The chances for an oocyte to cleave or for a pregnancy to continue decreased when the number of preovulatory follicles increased. The number of transferred embryos increased with oocyte cleavage rate in nonpregnant patients and in patients with an ongoing pregnancy, but such a relation was not found in the case of failed pregnancy (biochemical pregnancies and abortions). Failed pregnancies occurred in patients who demonstrated the highest oocyte cleavage rate (94.1%), compared with 81.2% for implantation failure and 84.8% for ongoing pregnancy (P less than 0.01). The cleavage rate was also higher in cases of multiple, compared with single, ongoing pregnancies after the transfer of three embryos (79.7% versus 64.5%, P less than 0.001). An important finding was the higher survival rate after triple-embryo transfer in patients, yielding numerous oocytes. The survival rate (fetuses per transferred embryos) after the transfer of three embryos was 14.9% in patients yielding five oocytes, versus 7.7% in patients yielding 3.4 oocytes (P less than 0.05). These results are related to oocyte and embryo viability and uterine ability for pregnancy.
The effect of maternal age on the incidence of chromosomally normal spontaneous abortion and different categories of chromosome abnormality among all clinically recognized human pregnancies was evaluated. The results provide no evidence for a significant association of age with sex chromosome monosomy or polyploidy, but clearly demonstrate an effect of age on the frequency of trisomy and chromosomally normal spontaneous abortions. Estimated maternal age-specific rates of trisomy among all recognized pregnancies were calculated and suggest that a majority of oocytes of women aged 40 years and older may be aneuploid.
Numerical chromosome abnormalities were studied in single blastomeres from arrested or otherwise morphologically abnormal human preimplantation embryos. A 6-h FISH procedure with fluorochrome-labeled DNA probes was developed to determine numerical abnormalities of chromosomes X, Y, and 18. The three chromosomes were stained and detected simultaneously in 571 blastomeres from 131 embryos. Successful analysis including biopsy, fixation, and FISH analysis was achieved in 86.5% of all blastomeres. The procedure described here offers a reliable alternative to sexing of embryos by PCR and allows simultaneous ploidy assessment. For the three chromosomes tested, numerical aberrations were found in 56.5% of the embryos. Most abnormal embryos were polyploid or mosaics, and 6.1% were aneuploid for gonosomes or chromosome 18. Extrapolation of these results to all human chromosomes suggests that the majority of abnormally developing and arrested human embryos carry numerical chromosome abnormalities.
During in-vitro fertilization (IVF) procedures, human preimplantation embryos were classified into four grades according to their morphological appearance under light microscopy. The grade IV group included poor quality embryos. In our IVF programme, these embryos were never transferred or frozen, and were thus available for cytogenetic analysis. Cytogenetic analysis was performed on 411 grade IV embryos from 327 couples participating in the IVF programme. A total of 118 embryos were successfully karyotyped using at least one metaphase. Normal diploid chromosomes were found in only 12 embryos, containing a total of 19 metaphases. All others (90%) showed abnormal or aberrant chromosome complements; 48 were aneuploid and six cases of single chromatids were noted; 14 embryos (11.8%) contained haploid complements, while the remaining 44 exhibited mosaics (2n/3n, n/2n, n/3n) or fragmented chromosome sets. Also, several structural aberrations and rearrangements were observed. These results indicate that the large majority of grade IV human embryos are chromosomally abnormal. This confirms the morphological assessment of the poor quality of these embryos and demonstrates the uselessness of both the transfer and the cryopreservation of grade IV embryos.
Attempts to increase the probability of a successful pregnancy in in-vitro fertilization (IVF) treatment by increasing the number of embryos transferred automatically also increase the probability of multiple pregnancies and their attendant risks. Even where the number of transferred embryos is limited to a maximum of three as in this and other centres, there is a high incidence of twins and triplets. The question therefore arises whether the number of transferred embryos should be further limited to a maximum of two in cases where the prognosis is otherwise good. The only objection to this idea is a possible lowering of pregnancy rate. The present study set out to investigate this question. No significant lowering of pregnancy rate was found, so that limiting the number of transferred embryos to two where the prognosis is otherwise good has now become standard practice in our centre. A good IVF prognosis was defined by the following criteria: first attempt for IVF, less than 37 years old, and good embryo development. From 183 patients fulfilling these criteria, 80 agreed to the transfer of two embryos (group 1) and 103 opted for a triple transfer (group 2). Patient characteristics and embryology results were similar in the two groups. In group 1, 34 patients (42.5%) became pregnant and in group 2, 50 (48.5%). This difference is not significant. Similarly, twin pregnancy rates in both groups were high; eight twin pregnancies (23.5%) in group 1 and 12 (24%) in group 2.(ABSTRACT TRUNCATED AT 250 WORDS)
To investigate the relationship between the embryo number and morphology in conception cycles and the incidence of multiple pregnancies.
The study is based on information received from a computerized data base.
In Vitro Fertilization Unit, Sapir Medical Center, Kfar Saba, Israel.
A total of 117 consecutive pregnancies resulted from replacement of fresh embryos in our IVF-ET program.
The impact of embryo quality, as assessed by morphological parameters, on the multiple pregnancy rate (PR).
Implantation rates positively correlated with the number and the quality of transferred embryos. However, no multiple pregnancies occurred when only two embryos were replaced. There were no multiple pregnancies when only embryos of low quality (grades 1 and 2) were transferred. Furthermore, there was no correlation between the number of replaced embryos of poor quality and the rate of implantation. The multiple PR increased from 10% when a mixture of high and low quality embryos were transferred to 30.76% when only embryos of highest quality were transferred.
The implantation rate of transferred embryos is directly correlated with the morphological scoring. The results of the study suggest that the number of embryos transferred should be balanced against their morphological quality to reduce the rate of multiple pregnancies.
The purpose of this study was to devise an embryo score to predict the likelihood of successful implantation after in-vitro fertilization (IVF). Unlike most studies dealing with the influence of embryo stage and morphology on pregnancy, our study was based on single rather than multiple embryo transfers. A total of 957 single embryo transfers were carried out. No delivery was obtained after any of the 99 transfers using 1-cell embryos or embryos obtained after delayed fertilization. In the remaining 858 transfers, the embryos had cleaved. Higher pregnancy rates were obtained with embryos displaying no irregular cells (11.7 versus 6.9%; P < 0.01) and embryos displaying no fragmentation (11.5 versus 8.1%; P < 0.05). The 4-cell embryos implanted 2-fold more often than embryos with more or less cells (15.6 versus 7.4%; P < 0.01). Based on these observations, we devised a 4-point embryo score in which embryos are assigned 1 point each if they (i) are cleaved, (ii) present no fragmentation, (iii) display no irregularities, and (iv) have four cells. Both pregnancy rate and take home baby rate were significantly correlated with embryo score. Each point of this score corresponds to a 4% increase in pregnancy rate. Interestingly, pregnancy rate was significantly lower in women aged > 38 years (8.2 versus 11.4%; P < 0.05), even though embryo quality was similar regardless of age. Single embryo transfer allowed us to define a simple and useful embryo score to choose the best embryo for transfer to optimize IVF and embryo transfer outcome. The use of this embryo score could decrease multiple pregnancies after multiple embryo transfers.
readers with a two-factor (blinding status and meta-analysis topic), weighted analysis of variance model with weights equal to the inverse of the variance of the in odds ratio for each meta-analysis performed by each team of readers.
A retrospective analysis of results from 114 initiated in-vitro fertilization cycles utilizing pronuclear embryo transfer is presented. Patients were unselected for age or infertility criteria, constituted a continuous series and were grouped according to response to stimulation (Group 1, ideal; Group 2, suboptimal) or ovarian reserve (Group 3, poor). At 16-18 h post-insemination, embryos were scored for alignment of pronuclei and nucleoli and the appearance of the cytoplasm, generating an embryo score (ES). Transfers were performed 24-26 h post-insemination using two to six embryos with the highest ES. A corrected score was calculated (total score/number of embryos; CS). A total of 114 initiated cycles resulted in 97 oocyte retrievals with 38 clinical pregnancies (39%; 15% implantation). Pregnancy rates were significantly different between the three groups; 37 pregnancies in Group 1 (55% clinical pregnancy; 20% implantation), none in Group 2 and one in Group 3 (6%; 2% implantation: P < 0.001). The ES of transferred embryos correlated with groups. There was a strong correlation between CS and implantation and delivery rates. CS >15 resulted in a 28% implantation; 65% delivery rate. CS <14 resulted in four pregnancies, one delivered. The data show that oocyte quality and pronuclear embryo morphology are related to implantation and that pronuclear embryos can be successfully selected for embryo transfer.
To determine if multinucleation in normally fertilized embryos is indicative of poor developmental or clinical pregnancy prognosis and to examine the ovulation induction characteristics associated with multinucleation.
Retrospective review.
A tertiary care institution.
Patients undergoing IVF-ET cycles (exclusive of other assisted reproductive technologies).
Cycles in which embryos had at least 1 multinucleated blastomere were compared with cycles in which all blastomeres exhibited no nucleus or a single nucleus (control).
When >50% of transferred embryos contained multinucleated blastomeres there was a significant reduction in implantation (3.4% vs. 14.7%), clinical pregnancy (9.1% vs. 29.1%), and live birth rates (7.5% vs. 27.6%) when compared with transfers of control embryos. In conjunction with this finding, multinucleate cycles had higher E2 levels and more follicles on the day of hCG administration, a higher number of oocytes retrieved, a higher fertilization rate, and more embryos transferred per patient than did the cycles that produced control embryos. When multinucleated embryos were present, but not transferred, the developmental capacity of the sibling embryo was reduced.
The evaluation of nuclear status using simple light microscopy is predictive of embryo developmental capacity and should be included in the embryo scoring system. The presence of multinucleated blastomeres in normally fertilized embryos is associated with a more effusive response to gonadotropin therapy and is indicative of a poor developmental outcome and lower clinical pregnancy rates.
In vitro fertilization is associated with a high risk of multiple births, which is a direct consequence of the number of embryos transferred. However, other factors that contribute to the risk are not well defined.
Using the data base established by the Human Fertilization and Embryology Authority in the United Kingdom, we studied the factors associated with an increased risk of multiple births in 44,236 cycles in 25,240 women. The factors included the woman's age, the cause and duration of infertility, previous attempts at in vitro fertilization, previous live births, number of eggs fertilized, and number of embryos transferred.
Older age, tubal infertility, longer duration of infertility, and a higher number of previous attempts at in vitro fertilization were all associated with a significantly decreased chance of a birth and of multiple births. Previous live birth was associated with an increased chance of a birth but not of multiple births. The higher the number of eggs fertilized, the higher the likelihood of a live birth. When more than four eggs were fertilized, there was no increase in the birth rate for women receiving three transferred embryos as compared with those receiving two, but there was a considerable increase in the rate of multiple births when three were transferred (odds ratio, 1.6; 95 percent confidence interval, 1.5 to 1.8).
Among women undergoing in vitro fertilization, the chances of a live birth are related to the number of eggs fertilized, presumably because of the greater selection of embryos for transfer. When more than four eggs are fertilized and available for transfer, the woman's chance of a birth is not diminished by transferring only two embryos. Transferring more embryos increases the risk of multiple births.
In eukaryotes, G2/M progression is mediated by activation of mitosis promoting factor (MPF). To ensure faithful chromosome segregation, the activity of key mitotic inducers and inhibitors are coupled with chromosome replication, spindle pole duplication, morphogenesis, and DNA damage. Evidence gathered in the past two years has underscored the importance of positioning MPF and its regulators in the proper place at the proper time to ensure orderly progression through the G2/M transition. Altering the spatial organization of G2/M regulators also contributes to prevention of mitosis following DNA damage.
To evaluate the effects of fragmentation and fragment removal in day 3 human embryos on implantation and pregnancy.
Retrospective analysis of ETs homogeneous with respect to embryo fragmentation.
A program of IVF-ET.
The study population consisted of 2,410 patients.
The degree and pattern of fragmentation were evaluated on days 2 and 3; microsurgical fragment removal was performed after assisted hatching on day 3.
Clinical pregnancy and implantation rates.
The degree and pattern of fragmentation significantly impact pregnancy and implantation. With the application of microsurgical fragment removal before ET, embryos with 6%-35% fragmentation implant with similar frequency. The presence of large fragments (type IV) is detrimental to the developing embryo, whereas localized or small and scattered fragments do not significantly affect implantation.
The potential of fragmented embryos for implantation is determined partly by the distribution of fragments. Adoption of an embryo classification system reflecting types of fragmentation is advisable. The use of microsurgical fragment removal significantly alters the course of development for some embryos and improves their implanting potential.
To analyze the incidence of numeric chromosomal abnormalities in preimplantation embryos from women with unexplained recurrent miscarriage (RM) so as to seek an etiology and to determine whether the use of IVF may be indicated to treat these cases.
Prospective controlled study.
University laboratory of reproductive genetics and a tertiary referral center for infertility.
Nine women with a mean (+/-SD) of 3.9 +/- 0.6 RMs who were undergoing IVF and preimplantation genetic diagnosis, and a control group of young (n = 10) and older (n = 6) patients who were undergoing preimplantation genetic diagnosis because of sex-linked diseases.
In vitro fertilization, embryo culture for 72 hours, blastomere biopsy, and analysis of chromosomes 13, 16, 18, 21, 22, X, and Y with the use of fluorescent in situ hybridization. Transfer of chromosomally normal embryos into the uterus.
Numeric chromosomal abnormalities in human embryos.
Sixty-six embryos from patients with RM were compared with 62 embryos from young patients and 41 embryos from older patients. There was a significant increase in the rate of abnormal embryos in the patients with RM and the older patients compared with the controls. Abnormalities in most of the chromosomes studied were higher in the RM group than in the control group, especially those affecting chromosome 13.
There was an increase in numeric chromosomal abnormalities in preimplantation embryos from women with RM that could be the cause of infertility in many couples with unexplained RM. The use of IVF in such circumstances may be indicated if successful preimplantation genetic diagnosis is added to the procedure.
Pregnancy and live birth rates following in-vitro fertilization decline rapidly with advancing maternal age partly because of an increase in age-related aneuploidies occurring in female meiosis. Screening oocytes or preimplantation embryos for common aneuploidies is now possible by polar body or cleavage stage biopsy and multicolour fluorescence in-situ hybridization.
To maximize birth rates, physicians who perform in vitro fertilization (IVF) often transfer multiple embryos, but this increases the multiple-birth risk. Live-birth and multiple-birth rates may vary by patient age and embryo quality. One marker for embryo quality is cryopreservation of extra embryos (if embryos are set aside for cryopreservation, higher quality embryos may have been available for transfer).
To examine associations between the number of embryos transferred during IVF and live-birth and multiple-birth rates stratified by maternal age and whether extra embryos were available (ie, extra embryos cryopreserved).
Retrospective cohort of 300 US clinics reporting IVF transfer procedures to the Centers for Disease Control and Prevention in 1996.
A total of 35554 IVF transfer procedures.
Live-birth and multiple-birth rates (percentage of live births that were multiple).
A total number of 9873 live births were reported (multiple births from 1 pregnancy were counted as 1 live birth). The number of embryos needed to achieve maximum live- birth rates varied by age and whether extra embryos were cryopreserved. Among women 20 to 29 years and 30 to 34 years of age, maximum live-birth rates (43 % and 36%, respectively) were achieved when 2 embryos were transferred and extra embryos were cryopreserved. Among women 35 years of age and older, live-birth rates were lower overall and regardless of whether embryos were cryopreserved, live-birth rates increased if more than 2 embryos were transferred. Multiple-birth rates varied by age and the number of embryos transferred, but not by whether embryos were cryopreserved. With 2 embryos transferred, multiple-birth rates were 22.7%, 19.7%, 11.6%, and 10.8% for women aged 20 to 29, 30 to 34, 35 to 39, and 40 to 44 years, respectively. Multiple-birth rates increased as high as 45.7% for women aged 20 to 29 years and 39.8% for women aged 30 to 34 years if 3 embryos were transferred. Among women aged 35 to 39 years, the multiple-birth rate was 29.4% if 3 embryos were transferred. Among women 40 to 44 years of age, the multiple-birth rate was less than 25% even if 5 embryos were transferred.
Based on these data, the risk of multiple births from IVF varies by maternal age and number of embryos transferred. Embryo quality was not related to multiple birth risk but was associated with increased live-birth rates when fewer embryos were transferred.
To evaluate the nonselective application of extended embryo culture on the outcome of IVF.
Retrospective analysis.
Private practice assisted reproductive technology center.
Seven hundred ninety nonselected patients undergoing IVF with controlled ovarian stimulation.
For day 3 ET, multicell embryos were cultured in human tubal fluid medium and 12% synthetic serum substitute. For day 5 ET, embryos were cultured for 48 hours in S1 medium and then for 48 hours in S2 medium.
Implantation rate (determined by total no. of visualized gestational sacs), ongoing pregnancy rate, and number of embryos available for ET.
Respective day 3 and day 5 implantation rates for patients aged <35 years (29.5% and 38.9%), patients aged 35-39 years (20.7% and 28.2%), and all patients combined (23.3% and 32.4%) were statistically significantly different. Significantly more embryos were transferred on day 3 than on day 5 for patients aged <35 years (2.9 vs. 2.4), patients aged 35-39 years (3.1 vs. 2.6), and all patients combined (3.0 vs. 2.5). The difference in ongoing pregnancy rates per retrieval was statistically significant for day 3 compared with day 5 transfers for all patients combined (35.9% vs. 43.8%). Cancellation rates for transfer after retrieval increased significantly for day 3 compared with day 5 transfer (2.9% vs 6.7%).
These results demonstrate the feasibility of using extended embryo culture in a nonselective manner for couples undergoing IVF. Overall, extended embryo culture was associated with a significant increase in pregnancy rates and implantation rates and a significant decrease in the number of embryos transferred. The rate of multiple implantation among patients aged <35 years warrants consideration of single blastocyst transfers for this group.
The German embryo protection law does not allow embryo selection, but only selection of pronuclear stage (PN) oocytes. Only
as many PN oocytes are allowed to be selected as are planned to be transferred. Therefore, a clinically applicable score to
assess the quality of PN oocytes would be helpful. A recently published score was used under the conditions of the German
embryo protection law in 74 non-selected, consecutive intracytoplasmic sperm injection cycles. Only criteria which could be
evaluated at the PN stage were included, i.e. not criteria which could only be assessed after pronuclear membrane breakdown
or the first cleavage division. Supernumerary PN oocytes were cryopreserved after selection. A mean PN score of <13 (sum of
scores of all selected PN oocytes/number of selected PN oocytes) led to a pregnancy rate of 4%, a mean PN score of ≥13 to
a pregnancy rate of 22%. Embryo morphology and cumulative PN were correlated (r = 0.52, P < 0.05). The negative predictive value was 92% at a threshold of 13 for the mean PN score. The use of this and perhaps additional
scoring systems of PN stage oocytes might help to offer patients in Germany the transfer of two selected PN oocytes, which
would reduce the multiple pregnancy rate.
Objective:
To select patients for day 3 vs. day 5 embryo transfer.
Design:
Retrospective analysis of assisted reproduction technology (ART) cycles comparing outcomes of day 3 and day 5 transfers.
Setting:
ART program of Brigham and Women's Hospital.
Patient(s):
Patients with day 3 or day 5 embryo transfers (n = 221 and 141, respectively).
Intervention(s):
Cycles with eight or more zygotes were stratified by the number of eight-cell embryos available on day 3 (none, one or two, or three or more).
Main outcome measure(s):
Number of blastocysts, implantation rates, ongoing pregnancy rates, and number of fetal heart beats.
Result(s):
With no eight-cell embryos on day 3, 0% and 33% pregnancies resulted from day 5 vs. day 3 transfers. With one or two eight-cell embryos on day 3, ongoing and high order multiple rates were not different between day 3 and day 5 transfers. With three or more eight-cell embryos, day 5 transfer resulted in a decrease in multiple gestations but no difference in ongoing pregnancy rates compared with day 3 transfer.
Conclusion(s):
With no eight-cell embryos on day 3, a day 3 transfer is warranted. With one or two eight-cell embryos, any benefit of day 5 transfer appears to be equivocal. With three or more eight-cell embryos, day 5 transfer is recommended.
Implantation involves complex molecular interactions between implanting blastocysts and the hormonally primed uterus. Gene targeting allows the generation of mice lacking a specific gene or genes and has proved to be of considerable value when combined with classical physiology in understanding many biological questions, such as the process of implantation. In this article, we review genes that have been demonstrated by gene targeting in mice to be required in the uterus for implantation. In particular, we focus on a specific class of developmental control genes, the mammalian Hox genes, and their role in this process. Lastly, we attempt to synthesize current knowledge about the genetic control of implantation and to build a working genetic model for the implantation pathway.
Successful implantation is the result of an intimate 'cross-talk' between the blastocyst and uterus in a temporal and cell-specific manner. Thus, both the uterine and embryonic events must be examined to better understand this process. Although various aspects and molecules associated with these events have been explored, a comprehensive understanding of the implantation process is still very limited. In this review, we have highlighted the importance of the blastocyst's activity state and the receptive state of the uterus in determining the 'window' of implantation. In this context, we provide a testable scheme that signifies the important roles of various key molecules in embryo-uterine interactions during implantation.
To determine the impact of blastocyst transfer on an oocyte donation program.
Retrospective review of embryo transfer in an IVF clinic.
Private assisted reproductive technology unit.
Two hundred and twenty nine patients undergoing oocyte donation.
Culture of pronucleate embryos to either day 3 or day 5 followed by embryo transfer.
Implantation rates, pregnancy rates, and multiple gestations were analyzed.
Implantation rates and pregnancy rates were significantly increased by moving to extended embryo culture and transfer on day 5. After day 3 transfers, implantation and pregnancy rates were 47.1% and 75%, respectively. In contrast, on day 5 these rates were increased to 65.8% and 87.6%. Concomitantly, there were significantly fewer embryos transferred on day 5 (2.1) compared to day 3 (3.2).
Blastocyst transfer is a highly effective treatment for patients who receive donor oocytes, allowing excellent pregnancy rates while significantly reducing the incidence of high-order multiple gestations.