Uterine position at real embryo transfer compared with mock embryo transfer

BACKGROUND: The purpose of this study was to determine the consistency in the uterine position between mock and real embryo transfer. METHODS: We reviewed 996 consecutive embryo transfer cycles (585 patients); 74% of patients had an anteverted (AV) uterus and 26% had a retroverted (RV) uterus at mock embryo transfer. All mock and real embryo transfers were performed under abdominal ultrasound guidance. RESULTS: Of 623 fresh embryo transfers in patients with an AV uterus at mock embryo transfer, only 2% became RV, while 55% of 213 embryo transfers in patients with an RV uterus on mock embryo transfer converted to AV at real embryo transfer (P < 0.0001). For frozen-thawed embryo transfer, 12% of AV uteri at mock embryo transfer became RV, while 33% of RV uteri became AV (P = 0.01). CONCLUSIONS: Our data suggest that an RV uterus at mock embryo transfer will often change position at real embryo transfer. Misdirecting the embryo transfer catheter can be avoided by accurate knowledge of the uterine position at the time of embryo transfer, which can be more accurately assessed by routine ultrasound guidance. Additionally, patients with an RV uterus at mock embryo transfer should still present with a full bladder for embryo transfer, since a significant number will convert to an AV position.     

Preliminary clinical experience with human blastocyst development in vitro without co-culture

This preliminary analysis was designed to quantify blastocyst development of supernumerary embryos without the use of feeder cells, conditioned medium or whole serum. Embryos derived from in-vitro fertilization (IVF) that were not transferred or cryopreserved were included in this study. Ova were harvested for IVF after a standard ovarian stimulation with gonadotrophin-releasing hormone agonist/ human menopausal gonadotrophin (GnRHa/HMG) or follicle-stimulating hormone (FSH). Ova were collected and culture in 150 microliters droplets of P1 medium under mineral oil, in groups at 37 degrees C under 5% CO2, 5% O2, 90% N2 (group A) or under 5% CO2 in air (group B) environment. Embryo transfer was performed 72 h post-harvest. Viable embryos not transferred or cryopreserved were placed in blastocyst medium and cultured for an additional 48 h in 5% CO2 in air. Embryos that exhibited an expanded blastocoelic cavity and well-defined inner cell mass at 120 h were counted. Of 838 supernumerary embryos cultured, 448 (53.5%) reached the expanded blastocyst stage by 120 h of culture. Patients were given the option of cryopreservation at that time. The embryos were cryopreserved using a standard protocol with serial addition of glycerol. Embryos reaching the blastocyst stage after more than 120 h of culture were not included. There was no difference in the proportions of blastocyst development between group A, 217/410 (53.5%) and group B, 231/428 (54%). To date, 16 patients have each had up to three thawed blastocysts transferred, out of whom seven became pregnant. This report demonstrates that a simple system of sequential culture generated acceptable, viable blastocyst development (54%) with supernumerary embryos, without the use of feeder cells, conditioned medium or whole serum. Recognizing the differential metabolic requirements of early and late cleavage stage embryos has enabled the application of a glucose/phosphate-free simple culture medium (P1) for up to 72 h of culture and a complex, glucose-containing medium (blastocyst medium) for subsequent blastocyst development.     

Limited value of morphological assessment at days 1 and 2 to predict blastocyst development potential: a prospective study based on 4042 embryos

BACKGROUND: Non-invasive and routine developmental markers are available to select the most viable embryo; however their respective values in terms of blastocyst development potential remain difficult to distinguish. METHODS: During this prospective study, the sequential growth of 4042 embryos individually cultured from day 1 to day 5/6 was recorded. Pronuclear morphology on day 1, and early cleavage, cell number and fragmentation rate on day 2 were evaluated for each zygote. Additionally, blastocyst transfers were analysed with regard to their implantation ability and early embryo development parameters. RESULTS: Once adjusted to each other, each of the four parameters remained related to blastocyst development. Early cleavage and cell number on day 2 were the most powerful parameters to predict the development of a good morphology blastocyst at day 5. Moreover, whereas transfers of a good morphology blastocyst were associated with high implantation and live birth rates, parameters of early development were not helpful in predicting their implantation ability. CONCLUSIONS: The combination of all four parameters allowed the prediction of blastocyst development with an area under the receiver operating characteristics curve of 0.688, which represents a fairly low prediction of embryo viability. Such results indicate that it is necessary to search for additional criteria, including the ability of the blastocyst to develop.     

The Graduated Embryo Score (GES) predicts blastocyst formation and pregnancy rate from cleavage-stage embryos

BACKGROUND: Embryo morphology and cleavage rates alone do not consistently identify embryos with high implantation potential following IVF. Blastocyst transfer has been reported to improve success rates by identifying potentially superior quality embryos. Algorithms for predicting IVF outcomes based on the presence of early developmental milestones have been proposed. Here we introduce the Graduated Embryo Score (GES). METHODS: Nucleolar alignment along the pronuclear axis, regular cleavage and degree of fragmentation at the first cell division, and cell number and morphology on day 3 were weighted to create a possible GES of 100 for each of 1245 fertilized embryos derived from 109 patients aged <40 years. The GES was correlated with IVF outcome. RESULTS: Of 983 embryos for extended culture, 349 (36%) developed to blastocyst and 180 (18%) were good quality (grade I-II). When ranked by cell number and morphology alone, 34% of embryos with > or =7 cells and <20% fragmentation formed good quality blastocysts. Using GES, embryos scoring 90-100 had 64% blastocyst formation compared with 31% scoring 70-85 and with 11% scoring 30-65. Embryos scoring 70-100 had 44% blastocyst development compared with 9% scoring 0-65. Fifty-six patients (51%) conceived on-going gestations from 294 transferred embryos. In patients with at least one transferred embryo scoring > or =70, the pregnancy rate was 59% compared with 34% if all embryos scored <70. The overall implantation rate was 28%. Among embryos scoring 70-100, an implantation rate of 39% was seen, compared with 24% among embryos scoring 0-65. CONCLUSIONS: Predicting which cleaved embryos will form blastocysts could permit the high success rates associated with blastocyst transfer to be achieved from day 3 embryo transfer.     

Uterine junctional zone contractions during assisted reproduction cycles. VIDEO

This study was designed to assess junctional zone contractions(JZ) during cycles of in-vitro fertilization (IVF) and embryotransfer in oocyte donors exposed to a long protocol regimefor ovarian stimulation. Real-time transvaginal ultrasound andadvanced audio-visual and computer technology were used to evaluatethe contraction pattern, frequency and velocity. At the timeof down-regulation JZ contractions were not observed. After7 days of superovulation all patients displayed cervico-fundal,fundo-cervical and random contractions. Cervico-fundal wavesdominated the picture at the time of human chorionic gonadotrophininjection. However, the activity was strongest on the day ofoocyte retrieval. At the time the percentage of opposing wavesincreased and fundo-cervical waves disappeared. The highestwave frequency and velocity (4.29±0.68 waves/min and2.73±0.54 mm/s respectively) were observed at the timeof oocyte retrieval. All patients had some JZ activity on days2, 3, and 4 after oocyte retrieval but regular wave-like contractilitygradually decreased and only single random movements were seenon day 4 after oocyte retrieval. In conclusion, JZ activitythroughout the IVF cycle is more exaggerated when compared tothe results reported from observations of the natural cyclebut follows a similar pattern. This fact can probably be explainedby the vastly different hormone levels. Higher JZ activity andcorrespondingly increased mobility of the endometrium may impairits receptivity and affect implantation.     

降钙素对体外培养的小鼠胚泡发育、分化的影响

目的：探讨降钙素(CT)与小鼠胚泡发育及植入的关系。方法：将孕D4小鼠胚泡随机分组并接种到添加不同成分的培养液内进行体外培养。于培养24、48h后在倒置显微镜下观察、记录胚泡的孵化、粘附及扩展生长情况。结果：培养24h后,添加10nmol/L CT组与联合培养组胚泡的粘附率分别为31．71％、32．35％,和对照组(16．30％)相比,均有明显增加。培养48h后,添加10nmol/L CT组与联合培养组胚泡的孵化率、粘附率、扩展率均比对照组有非常显著增高。结论：CT可明显地促进体外培养的小鼠胚泡孵化、粘附及扩展生长,与植入过程密切相关。     

The effect of pronuclear morphology on embryo quality parameters and blastocyst transfer outcome.

BACKGROUND: Embryo quality may be accurately assessed as early as the pronuclear zygote phase, as shown in recent studies. However, it is not known whether good quality zygotes are destined to become good quality cleavage stage embryos and blastocysts. METHODS: In this retrospective study, 86 intracytoplasmic sperm injection-embryo transfer cycles were studied where each available embryo was scored from the zygote until the blastocyst stage. Embryonic normality parameters such as pronuclear pattern, early cleavage, cleavage stage embryo grade, the presence of embryos with > or =8 cells on day 3 and blastocyst quality were recorded. Embryo transfer was undertaken at the blastocyst stage and the outcome was studied according to the pronuclear pattern exhibited by the zygotes. RESULTS: Embryos that showed an ideal pronuclear pattern (0 PN pattern) cleaved earlier and faster and resulted in better quality cleavage stage embryos and blastocysts. The incidence of blastocyst formation was 72% in zygotes showing a 0 PN pattern, compared with 12.7% in zygotes with double pronuclear abnormality. Higher implantation and pregnancy rates were obtained when at least one blastocyst derived from a 0 PN pattern zygote was included in the set of embryos to be transferred. CONCLUSIONS: Our results indicate that the pronuclear pattern of the zygote is closely related to blastocyst formation and quality. Blastocysts derived from 0 PN zygotes have a higher potential for implantation.     

Day 3 embryo transfer with combined evaluation at the pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer

BACKGROUND: The respective advantages of day 3 and day 5 embryo transfer are a matter of debate. Previous comparisons did not include pronuclear stage zygote scoring and cumulative success rates (fresh and cryopreserved embryos). METHODS: Patients were randomized prospectively for day 3 or day 5 embryo transfer. Day 3 embryos were selected for transfer and cryopreservation by using combined evaluation at the pronuclear and cleavage stages. RESULTS: There was no difference between day 3 and day 5 fresh embryo transfers as to the rates of pregnancy (58 versus 62%), clinical pregnancy (56 versus 58%), delivery (50 versus 48%), implantation (35 versus 38%) and birth (33 versus 36%) rates. The corresponding values for cryopreserved embryo transfers were also similar. However, day 3 embryo transfer compared favourably with day 5 transfer when the pregnancy (90 versus 66%), clinical pregnancy (85 versus 62%) and delivery (77 versus 52%) rates were calculated per oocyte recovery attempt. CONCLUSIONS: With a selected population of good prognosis patients and our embryo selection criteria, the implantation potential of day 3 and day 5 embryos is equal. Per oocyte recovery attempt, day 3 transfer is more clinically efficient than day 5 transfer, but at least one transfer of cryopreserved embryos is necessary to manifest this superiority.     

An in-vitro model for stromal invasion during implantation of the human blastocyst

BACKGROUND: Implantation failure is likely to be a major cause of infertility. Studies in mice have identified a number of molecules that are involved in implantation, but the mechanisms of implantation in the human remain unclear, largely due to the lack of models for implantation in the human that provide functional information. METHODS: Human hatched blastocysts were co-cultured with human endometrial stromal cell monolayers. Time-lapse photography of implanting blastocysts, immunostaining for cytokeratin and actin, and measurement of hCG secreted into the culture supernatants were performed. RESULTS: Blastocysts attached to and implanted into the stromal cell layer. Trophoblast outgrowth onto, and invasion into, the stromal cell layer occurred largely at two opposite poles, the orientation of which was aligned to that of the stromal cell fibroblasts. High-resolution image analysis demonstrated that the trophoblast completely penetrated the stromal cell layer. Immunostaining of whole-mounts of implantation sites revealed distinctive actin and cytokeratin-positive anchoring structures adjacent to the basal surface of the trophoblast. Blastocysts implanting into stromal cells secreted higher levels of hCG compared with those cultured on plastic. CONCLUSIONS: A robust model for the study of mechanisms of implantation of the human embryo into the endometrial stroma has been established.     

Granulocyte-macrophage colony-stimulating factor promotes human blastocyst development in vitro

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is synthesized in the female reproductive tract and has been implicated in the growth and development of the preimplantation embryo in rodent and livestock species. To examine the effect of GM-CSF on human embryo development in vitro, surplus frozen 2-4-cell embryos were cultured in media supplemented with 2 ng/ml recombinant human GM-CSF. The addition of cytokine increased the proportion of embryos that developed to the blastocyst stage from 30 to 76%. The developmental competence of these blastocysts, as assessed by hatching and attachment to extracellular matrix-coated culture dishes, was also improved by GM-CSF. The period in culture required for 50% of the total number of blastocysts to form was reduced by 14 h, and blastocysts grown in GM-CSF were found to contain approximately 35% more cells, due primarily to an increase in the size of the inner cell mass. The beneficial effect of GM-CSF was exerted in each of two sequential media systems (IVF-50/S2 and G1. 2/G2.2) and was independent of the formulation of recombinant cytokine that was used. These data indicate that GM-CSF may have a physiological role in promoting the development of the human embryo as it traverses the reproductive tract in vivo, and suggest that addition of this cytokine to embryo culture media may improve the yield of implantation-competent blastocysts in human in-vitro fertilization programmes.     

Transcriptome analysis in blastocyst hatching by cDNA microarray

BACKGROUND: Hatching is an important process for early embryo development, differentiation and implantation. However, little is known about its regulatory mechanisms. By integrating the technologies of RNA amplification and cDNA microarrays, it has become possible to study the gene expression profile at this critical stage. METHODS: Pre-hatched and hatched ICR mouse embryos (25 blastocysts in each group were used in the triplicate experiments) were collected for RNA extraction, amplification, and microarray analysis (the mouse cDNA microarray, 6144 genes, including expressed sequence tags). RESULTS: According to cDNA microarray data, we have identified 85 genes that were expressed at a higher level in hatched blastocyst than in pre-hatched blastocysts. In this study, 47 hatching-related candidate genes were verified via re-sequencing. Some of these genes have been selected and confirmed by real-time quantitative RT-PCR. These hatching-specific genes were also expressed at a lower level in the delayed growth embryos (morula or blastocyst without hatching at day 6 post hCG). These genes included: cell adhesion and migration molecules [E-cadherin, neuronal cell adhesion molecule (NCAM), lectin, galactose binding, soluble 7 (Lgals7), vanin 3 and biglycan], epigenetic regulators (Dnmt1, and SIN3 yeast homolog A), stress response regulators (heme oxygenase 1) and immunoresponse regulators [interleukin (IL)-2-inducible T-cell kinase, IL-4R, interferon-gamma receptor 2, and neurotrophin]. The immunostaining of E-cadherin and NCAM showed strong and specific localization in hatched blastocyst. CONCLUSIONS: This work provides important information for studying the mechanisms of blastocyst hatching and implantation. These hatching-specific genes may have potential as new drug targets for controlling fertility.     

Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage

BACKGROUND: A correlation between morphology, developmental competence and chromosome abnormalities is established. However, since absolute correlations are rare, embryo selection remains one of the most arduous tasks in assisted reproduction. This study was undertaken in order to determine which chromosomal abnormalities are compatible with development to the blastocyst stage. METHODS: Embryos diagnosed by preimplantation genetic diagnosis (PGD) as chromosomally abnormal or unsuitable for transfer were cultured to day 5 or 6. Morphology and development were observed daily. After extended culture, embryos were fixed and analysed by two rounds of FISH with the same probes used for PGD. RESULTS: Some types of numerical chromosome abnormalities do not preclude full differentiation in vitro. For instance, extensive mosaicism was detected in blastocysts and trisomic embryos reached the blastocyst stage with a frequency of 37%. Interestingly, only those monosomies compatible with first trimester development (monosomy X and 21) were detected at blastocyst stage. CONCLUSION: Even though there is a strong selection against chromosomally abnormal embryos, extended culture to day 5 or 6 cannot be used as a reliable tool to select against clinically relevant chromosome abnormalities such as trisomies.     

A prospective trial of blastocyst culture and transfer

BACKGROUND: The aim of this study was to evaluate the effectiveness of the blastocyst culture method compared with the conventional day 3 transfer method using a prospective trial. METHODS: A total of 235 patients with 273 cycles were evaluated for a period of almost 1 year. Depending upon the sequence in which the ovum retrieval was performed, patients were prospectively assigned (alternate allocation) to a culture period of 3 or 5 days duration. Embryos were transferred either on day 3 (after culture in human tubal fluid) or on day 5 (after culture in sequential media). RESULTS: The pregnancy rates after embryo transfer on days 3 and 5 were similar at 26.5 and 25.9% respectively. Among the day 5 embryo transfer group, patients were divided into three groups corresponding to three sequential media. The pregnancy rates were 32.0% using Irvine blastocyst medium, 6.9% using G1.2/G2.2 and 32.4% using Cook blastocyst medium. CONCLUSIONS: The results of our study were not as successful as previous studies had been. Additionally, there may have been problems in day 5 embryo transfer, such as choosing the sequential media, quality control, contamination and so on. From the results of this study, it appears that day 5 embryo transfer has no advantages for ordinary patients of assisted reproductive technology.     

Hydrosalpinx fluid does not adversely affect the normal development of human embryos and implantation in vitro

Several retrospectively designed studies have shown an associationbetween the presence of hydrosalpinx and impaired implantationand pregnancy rates among in-vitro fertilization (IVF) patients.In the present study we have evaluated the influence of hydrosalpinxfluid on normal human embryo development and implantation. Surplus,donated frozen embryos (n = 183) from IVF patients were usedto study the effects on blastocyst development of hydrosalpinxfluid at concentrations of 50 and 100% compared with controlsin S2 medium. The fluids were analysed for concentrations ofelectrolytes, osmolarity, protein content, endotoxin levels,bacterial or fungal contamination, pH and haemoglobin content.There was no difference in blastocyst development in culturesunder mineral oil when control cultures (15/42 = 36%) were comparedwith cultures in 50% hydrosalpinx fluid (32/96 = 33%). The onlybiochemical parameter which correlated with capacity for blastocystdevelopment was pH in hydrosalpinx fluid/medium (50/50%) afterequilibration in 5% CO₂ in air. When embryos were cultured in100% hydrosalpinx fluid the blastocyst development was 14% (5/36)in comparison to control 33% (3/9). The original experimentwas repeated in an open culture system without the protectionof mineral oil but still in the presence of 50% hydrosalpinxfluid. The rate of blastocyst development was within the samerange in the open system. In three separate experiments, thecapability of expanded blastocyst to implant on multilayer artificialendometrium was tested. In these experiments, 1/3, 4/5 and 9/9blastocysts implanted. The present study demonstrates that hydrosalpinxfluid does not generally exert any major negative effects onin-vitro development of human embryos or on the implantationprocess in vitro.     

Symposium: reproduction in baboons. From blastocyst to placenta: the morphology of implantation in the baboon

Implantation and placentation in the baboons share many morphologicalfeatures with other primates, as well as having some specificdistinctions. The ability to use deturgescence of the sex skinas a method of timing ovulation and the ease with which theuterine lumen can be flushed have been used to examine morphologicalaspects of blastocyst differentiation and implantation in thisspecies. Preimplantation blastocysts were obtained by non-surgicalflushing of the uterus 68 days after ovulation, and implantationsites were excised from uteri removed on days 10-16 of gestation.All tissues were prepared for electron microscopy by aldehydefixation and plastic embedding. Maturation of trophoblast fromthe compacted morula stage to the expanded blastocyst stageincludes increase in numbers of polyribosomes, changes in conformationof mitochondria, and development of an effective endocytic apparatus.An endodermal layer forms beneath the inner cell mass priorto loss of the zona pellucida, and parietal endodermal cellsextend beyond the inner cell mass. Azonal blastocysts have regionsof syncytial trophoblast adjacent to the inner cell mass, andthey may represent adhesion stages of early implantation. Inearly postimplantation stages, trophoblast replaces the uterineepithelium and processes of syncytial trophoblast invade dilatedsuperficial maternal vessels. In subsequent lacunar stages thereis rapid elevation of the developing conceptus above the uterinesurface as the lacunae enlarge. Cytotrophoblast rapidly entersmaternal vessels, and arterioles are partially or completelyoccluded by migrating cytotrophoblast. The early access to controlledmaternal blood flow apparently allows trophoblastic lacunaeto expand superficially as opposed to more extensive endometrialinvasion.     

In-vivo blastocyst production and ovum yield among fertile women

We evaluated ova recovered from 13 fertile women undergoinguterine lavage for purposes of embryo donation, and assesseddifferences in blastocyst production and ovum yield among subjects.Six women produced 10 blastocysts during 31 insemination cycles(32%). Yet, despite undergoing at least four insemination cyclesapiece, seven women produced no blastocysts in 52 lavage attempts.Comparing tbe ovum recovery rates between the blastocyst-producingand non-blastocyst-producing groups, we found the former morelikely to yield ova (P 0.05), and more importantly 10 of 20recovered ova were blastocysts. We conclude that donor fecundityis highly variable among fertile women, and that reproductivehistory alone does not predict ovum donor efficiency.     

Implantation: Uterine fluid human decidua-associated protein 200 and implantation after embryo transfer

Uterine fluid samples from 109 patients undergoing in-vitrofertilization and embryo transfer were obtained so as to examinethe relationship between the uterine fluid concentration ofhuman decidua-associated protein (hDP) 200 and the implantationrate. The sampling was performed on the day of embryo transferwith a Wallace catheter, used for the testing of cervical patencybefore embryo replacement. The implantation rate, as well asthe pregnancy rate, demonstrated a significantly positive correlationwith the concentration of hDP 200 in the uterine fluid, measuredjust before embryo transfer. These results indicate that hDP200, identified as a rheumatoid factor secreted by the endometrium,may be involved in the implantation process.     

Blastomere development after embryo biopsy: a new model to predict embryo development and to select for transfer

One of the most important and unsolved problems in in-vitro fertilization is to decide which embryos are more suitable to implant and therefore should be transferred. We analysed the in-vitro development of isolated biopsied blastomeres and compared it to the development of the original embryo, in order to find a relationship that could show the embryo's potential future development and so increase implantation rates. A total of 66 normally fertilized human embryos were biopsied at the 6- to 10-cell stages. At day 6, blastomeres were counted by nuclear labelling. A total of 33 embryos (50%) reached the blastocyst stage. Of the isolated blastomeres, 63% divided and 53% cavitated over 3 days in culture. Of the blastomeres taken from embryos that developed to the blastocyst stage, 88% divided, 79% cavitated, 76% divided and cavitated and 9% neither divided nor cavitated. In those from arrested embryos, 39% divided (P < 0.001), 21% cavitated (P < 0.001), 15% divided and cavitated (P < 0.001) and 55% neither divided nor cavitated (P < 0.001). Blastomeres biopsied from embryos that reached the blastocyst stage showed a significantly higher proportion of division and cavitation than those originated from arrested embryos. Culture of the isolated blastomeres can demonstrate those embryos more likely to develop to the blastocyst stage and that are probably more suitable to implant. Cryopreserving biopsed embryos and culturing blastomeres would increase implantation rates. Embryos can then be selected according to the blastomere development and thawed for transfer in a future cycle.     

Monozygotic twins and transfer at the blastocyst stage after ICSI

The incidence of monozygotic twinning (MZT) is higher in pregnancies conceived after assisted reproduction than after natural conception. Alterations, produced by ovarian stimulation, in-vitro culture conditions and specifically alterations of zona pellucida are mentioned as possible causes of this phenomenon. A retrospective review was performed of the incidence of MZT in pregnancies generated in our centre during the period of January 1996 to December 1999. This variable was compared in 129 gestations that resulted from blastocyst transfer (occurring from September 1998 to August 1999) with 814 pregnancies produced by transfers of 4- to 8-cell embryos. Follicular development was induced with human menopausal gonadotrophin and urinary FSH during 1996 and 1997 and with recombinant FSH during 1998 and 1999. Blastocysts were cultured in sequential media using S1 or G1 up to 72 h and S2 or G2 to day 5. Five of the 129 pregnancies generated by blastocyst transfers were complicated by MZT gestation (3.9%). In comparison, only six of 814 pregnancies occurred from 4- to 8-cell transfers (0.7%), a difference that is statistically significant (P< 0.001 with Yates correction). The results confirm an increase of MZT in pregnancies from intracytoplasmic sperm injection as compared to the natural incidence. Moreover, the frequency of MZT was significantly higher when transfers were performed at the blastocyst stage, suggesting that extended in-vitro culture of embryos may be associated with alterations of the zona pellucida and the hatching process.