A method using the chick embryo amnion for mammalian embryo culture

Summary A procedure has been developed for incubating early stage mammalian embryos within the early chick amniotic cavity. Normal embryonic growth and development following extended culture in the chick embryo amnion has been verified by the live birth of transferred caprine embryos. This method is a simple and inexpensive alternative to in vitro and in vivo methods currently used for culturing mammalian embryos prior to transfer.     

苄普地尔对小鼠的致畸作用

苄普地尔分５０ｍｇ／ｋｇ、１００ｍｇ／ｋｇ、３００ｍｇ／ｋｇ三个剂量。小鼠分着床期试验组和器官形成期试验组。结果显示：着床期试验组孕鼠体重增长，着床数，活胎数，吸收胎数，死胎数无明显改变。     

Decisions for the fate of frozen embryos: fresh insights into patients' thinking and their rationales for donating or discarding embryos

BACKGROUND: In the final decision for the disposition of unused IVF embryos patients must choose between options involving either donation or destruction, and this decision must be made in a context where there is tension about the status of embryos (i.e. whether viewed as potential children or as a base for further development) and whether embryo donation is adoption or tissue donation. This study explored the emotive experience of making a decision for either the destruction or donation of unused embryos. METHODS: Thirty-three patients (9 women and 12 couples) who discarded embryos and 15 (7 women and 4 couples) who donated embryos were interviewed. Interview data were analysed with particular attention to elements of moral deliberation and use of analogy. RESULTS: Adoption and tissue donation metaphors were both identified, and further, a metaphor of pregnancy termination was identified and found to be highly influential in the decision to donate embryos. Contrary to the majority of current evidence, this study found that participants who discarded embryos emphasized the adoption metaphor while embryo donors emphasized the metaphor of pregnancy termination. For each group the decision was driven by awareness of the option they did not want. CONCLUSIONS: The pregnancy termination metaphor emerged as morally relevant and this holds implications for defining and discussing embryo discard in counselling and consent processes.     

The predictive value of using a combined Z-score and day 3 embryo morphology score in the assessment of embryo survival on day 5

BACKGROUND: The purpose of this study was to evaluate the ability of using the Z-score alone, or, in combination with the day 3 embryo morphology score, to predict embryo viability at day 5 from a large cohort of embryos derived from patients undergoing treatment with IVF/ICSI. METHODS AND RESULTS: In this retrospective study, a total of 1894 zygotes from 346 treatment cycles (295 couples) was analysed between January 2001 and May 2002. The Z-scoring system was useful in predicting day 5 embryo survival. The mean +/- SD day 5 embryo survival rates were 78.2 +/- 1.7, 49.0 +/- 2.5, 21.4 +/- 3.2 and 11.8 +/- 5.6% for Z-1, Z-2, Z-3 and Z-4 zygotes groups respectively. Embryos derived from Z-1 scores and grade I day 3 embryo scores showed the best day 5 embryo survival and a very high implantation potential. CONCLUSIONS: These data suggest that a combined evaluation of the Z-score and day 3 embryo morphology is highly predictive of embryo outcome after IVF/ICSI. The Z-score could be of great help in the selection of embryos for cultures extended to later stages. The Z-score alone, or preferably in combination with day 3 embryo morphology, is useful in the determination of the most suitable embryos and the number of embryos for transfer, thus achieving the optimal chance of conception while reducing the risk of high order multiple pregnancy.     

钢丝环法胚胎玻璃化冷冻技术的初步应用

目的探索自制钢丝环用于人卵裂胚及囊胚玻璃化冷冻的效果。方法来源于生殖中心IVF或ICSI助孕的共33个胚胎（卵裂胚20个,囊胚13个）,用自制钢丝环作为胚胎载荷物行玻璃化冷冻,复苏和移植后,观察其复苏存活率、临床妊娠率、植入率和流产率。结果卵裂胚和囊胚的复苏率、植入率和妊娠率分别为85%、23.5%、28.6%（2/7）和76.9%、40%、33.3%（2/6）,无流产病例。结论使用自制钢丝环作为胚胎载荷物行玻璃化冷冻,对人类卵裂胚和囊胚均可获得良好的临床效果,可以作为常规冷冻的一种应急或补充技术。     

How to facilitate decisions about surplus embryos: patients' views

BACKGROUND: In Victoria, Australia, legislation governing fertility treatment provides that surplus human embryos must not be stored for longer than 5 years. Couples must then choose one of three options: discard, donate to research or donate to another infertile couple. Previous research suggests that many people find these decisions difficult and emotionally distressing. This study aims to elucidate the nature of these difficulties and to identify ways in which the decision-making process could be facilitated. METHODS: This project used a combination of qualitative research methods. In total, 42 people agreed to participate in either a structured interview or a group discussion. All participants had completed IVF treatment and had surplus embryos in storage. The aim of the interviews was to discuss participants' decision making regarding their surplus embryos. Data were thematically analysed. RESULTS: Most participants described the decision-making process as difficult and emotional. Findings indicate that participants could be assisted by more information about each of their current options, and opportunities to talk to others in similar situations. Many responded positively to the idea of having more options, including choice about which research projects to donate to (directed research), and about the recipients of their donated embryos (directed donation). CONCLUSIONS: This study suggests that there are practical ways to assist people in making decisions about their surplus embryos, which could be easily implemented. In addition, the study demonstrated interest in the possibility of directed donation to other couples.     

High outcome predictability after IVF using a combined score for zygote and embryo morphology and growth rate

BACKGROUND: A scoring system has been developed to determine preimplantation embryo quality, and used to select embryos for transfer into the uterus of patients. METHODS AND RESULTS: The system was used to study early embryo development and to test whether these scores alone can accurately predict IVF outcome. Following zygote and embryo scores through early development, the data showed that a top quality zygote does not necessarily indicate that the resulting embryo will be top quality after in-vitro culture. The embryo quality score can change dramatically when embryos are cultured to day 2 or 3 post-fertilization. Pregnancy rates and implantation rates were compared with the cumulative and separated zygote and embryo scores. Analysis of the predictability of scoring systems suggested that morphological scores alone are relatively unpredictive of IVF outcome. When weighted for in-vitro growth rate, scores were highly predictive, more so than the rate of development alone. CONCLUSIONS: These data suggested that a combination of in-vitro growth rate and morphological analysis both of zygotes and embryos was highly indicative of outcome after IVF. The results can be adopted to the single embryo transfer approach to IVF.     

Pregnancy achieved by transferring blastocysts into endometrial stroma in mice.

To prevent the extra-uterine discharge of transferred embryos, we directly inserted mouse embryos into the endometrial stroma (intra-endometrial embryo transfer). A 27G injection needle was inserted near the utero-tubal junction into the endometrial stroma. After removal of the needle, a glass micropipette was inserted and one embryo was transferred with a very small amount of culture medium. To determine the feasibility of this method, the uterine lumen was flushed with phosphate-buffered saline from the tubal ends immediately after transferring blastocysts into pseudopregnant mice on day 2 and day 4. The rates of recovery of embryos from the uterine lumen were 5.0% (1/20; day 4) and 15.0% (3/20; day 2). These results suggest that a high rate of intra-endometrial embryo transfer is possible. The embryonic viability rates (number of viable grown fetuses/number of blastocysts transferred) of this method were 50.0% (28/56; day 4) and 25.0% (5/20; day 2). Living offspring were delivered from both recipients which had received embryos on day 2 and day 4 of pseudopregnancy. In human in-vitro fertilization and embryo transfer, attempts have also been made to immobilize the embryos, and this method might be clinically applicable. Moreover, this method will be a good in-vivo model for studies on the mechanism of implantation.     

Implantation: Two instead of three embryo transfer in in-vitro fertilization

The rational of transferring two instead of three embryos wasstudied through 468 in-vitro fertilization (IVF) treatment cyclesin 287 couples. The quality of 1224 embryos was determined accordingto the fragmentation rate and the morphology as good (A) andpoor (B). The influence of the number of embryos transferred(two or three) on the pregnancy rate when the same quality orcombinations of good and poor quality embryos transferred wasexamined. When only good quality embryos were transferred thepregnancy rates in double (AA) and triple (AAA) embryo transferwere 40.5 (17/42) and 42.9% (30/70) respectively (not significant).When only poor quality embryos were transferred, the pregnancyrates in double (BB) and triple (BBB) embryo transfers were11.0% (11/ 100) and 22.9% (16/70) respectively (P < 0.001).On the other hand, when good and poor quality embryos were transferredtogether as AB in double and as AAB and ABB in triple embryotransfer, the pregnancy rates were 36.8 (14/38) and 39.9% (59/148)respectively (not significant). There was no difference in themiscarriage rate between double and triple embryo transfers;16.7 and 18.1% respectively. The multiple pregnancy rate was14.3% for double embryo transfers and 32.4% for triple embryotransfers (P < 0.001). This study demonstrates that if thereis at least one good quality embryo available for transfer,then double instead of triple embryo transfer will not yielda significantly lower pregnancy rate. The influence of the numberof embryos transferred on the pregnancy rate became significantwhen only poor quality embryos were transferred. In conclusion,as long as at least one good quality embryo is available fortransfer, we may consider the transfer of double instead oftriple embryos.     

Implantation: Trophectoderm projections: a potential means for locomotion, attachment and implantation of bovine, equine and human blastocysts

The behaviour of bovine, equine and human blastocysts was studiedin vitro by time-lapse videomicrography and computer imaging.This study revealed that cytoplasmic extensions of the trophectoderm[‘trophectoderm projections’ (TEP)] were expressedby embryos of all three species, prior to or during zona escape.Bovine and human blastocysts escaped their zonae with a combinationof blastocoele expansion, collapse and re-expansion coupledwith the penetration of the zona pellucida by TEP. In equineembryos, after several cycles of blastocoele expansion and collapse,trophectoderm ruptured the zona with the concomitant appearanceof TEP. This study provides documentation that TEP are expressedby a diverse range of mammalian species, bringing the totalnumber of species in which this phenomenon is found to six,since TEP are also known to be expressed by guinea-pig, hamsterand rhesus monkey blastocysts, representing rodents, ungulatesand primates. In all species studied, the dynamic nature (extension,retraction, and angular movement) of the TEP was similar, movingin an undulating manner with rapid cycles of extension and retraction.Because TEP appear to be a genera] feature of mammalian blastocysts,they are implicated in one or more key events in early development,namely zona escape, attachment and/or implantation.     

An analysis of the effect of the PMSG-HCG interval on the two-cell block

The effect of increasing the interval between the administrationsof pregnant mare's serum gonadotrophin and HCG to female micefrom 48 to 60 h on the mating frequency, ovulation rate, fertilizationrate and development through the twocell stage has been investigated.It is concluded that use of the longer interval is of no advantage,and may be disadvantageous     

Human preimplantation embryo cryopreservation: selected aspects

This report describes the results of cryopreserving human preimplantation zygotes and cleaved embryos (2-4 cells) in our in-vitro fertilization programme. Cryopreserved zygotes and cleaved embryos resulted in similar post-thaw survival rates (74.8 versus 70.9%). Pregnancy rates per retrieval cycle (RC) and embryos transferred per pregnancy for frozen-thawed zygotes versus frozen-thawed cleaved embryos were 21.8 versus 11.5% (P less than 0.2) and 12.6 versus 17.5 (P less than 0.2), respectively. Pregnancy rates increased significantly for both fresh (P less than 0.0005) and frozen-thawed (P less than 0.05) embryos as the number of embryos replaced per transfer increased from one to three or more. Frozen-thawed embryos resulted in multiple implantation rates per transfer of 25 compared to 6.4% (P less than 0.1) for fresh embryos when two embryos were replaced. Pregnancy rates were reduced for fresh (P less than 0.05) and frozen-thawed (P less than 0.1) embryos obtained from patient retrieval cycle numbers greater than 3. The method of follicular stimulation during the retrieval cycle did not affect frozen-thawed embryo survival rates. There was no difference in pregnancy rates from frozen-thawed embryos replaced during natural or clomiphene citrate transfer cycles. Patients with cryopreserved embryos had cumulative pregnancy rates of 37.1% (66/178) compared to 23.5% (110/468) (P less than 0.01) for patients with no embryos cryopreserved; cryopreservation of preimplantation embryos is a reliable therapeutic procedure that enhances achievement of pregnancy through in-vitro fertilization.     

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.     

Implantation: Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers

The purpose of this study was to devise an embryo score to predictthe likelihood of successful implantation after in-vitro fertilization(IVF). Unlike most studies dealing with the influence of embryostage and morphology on pregnancy, our study was based on singlerather than multiple embryo transfers. A total of 957 singleembryo transfers were carried out. No delivery was obtainedafter any of the 99 transfers using 1-cell embryos or embryosobtained after delayed fertilization. In the remaining 858 transfers,the embryos had cleaved. Higher pregnancy rates were obtainedwith embryos displaying no irregular cells (11.7 versus 6.9%;P < 0.01) and embryos displaying no fragmentation (11.5 versus8.1%; P < 0.05). The 4-cell embryos implanted 2-fold moreoften than embryos with more or less cells (15.6 versus 7.4%;P < 0.01). Based on these observations, we devised a 4-pointembryo score in which embryos are assigned 1 point each if they(i) are cleaved, (ii) present no fragmentation, (iii) displayno irregularities, and (iv) have four cells. Both pregnancyrate and take home baby rate were significantly correlated withembryo score. Each point of this score corresponds to a 4% increasein pregnancy rate. Interestingly, pregnancy rate was significantlylower in women aged >38 years (8.2 versus 11.4%; P < 0.05),even though embryo quality was similar regardless of age. Singleembryo transfer allowed us to define a simple and useful embryoscore to choose the best embryo for transfer to optimize IVFand embryo transfer outcome. The use of this embryo score coulddecrease multiple pregnancies after multiple embryo transfers.     

Ultrastructure of a two-cell human embryo

Summary A two-cell human embryo recovered from the Fallopian tube 82 h following the LH peak in plasma and 37 h after a single episode of intercourse was examined by transmission electron microscopy. At the time of recovery the embryo was denuded of cumulus cells, and both the zona pellucida and the two adjoining blastomeres were intact. The finding of two polar bodies in the perivitelline space, two nucleated blastomeres and remnants of the fertilizing sperm tail within the cytoplasm of one of them, were considered as evidences that the embryo was normally fertilized. Among the most compicuous features found were the presence of very distinct desmosome-like structure between blastomeres, and the cytoplasmic cell organelles distribution in three areas referred as: a sub-cortical, a middle and a perinuclear bands. An outstanding feature was the extensive blebbing of the nuclear envelope. In general, the features seem to correspond to a normally developing two-cell embryo undergoing cleavage at a normal rate.     

Impact of patients' choice for single embryo transfer of a top quality embryo versus double embryo transfer in the first IVF/ICSI cycle

BACKGROUND: The aim of this study was to evaluate the impact of transferring a single top quality embryo in the first IVF/ICSI cycle of patients <38 years old who chose to have one or two embryos transferred. METHODS: A total of 262 patients participated in the study, and 243 transfers were performed: 156 (64%) patients chose the transfer of a single top quality embryo, if available, and two non-top quality embryos if not available; 87 (36%) patients chose to have a double embryo transfer regardless of embryo quality. RESULTS: In the first group an ongoing pregnancy rate of 40% (63/156) with a twin pregnancy rate of 2% (1/63) was achieved. In the second group the ongoing pregnancy rate was 44% (38/87) with 26% (10/38) twin pregnancies. In the patient group with only one embryo transferred, irrespective of the patient's choice, the ongoing pregnancy rate was 43% (54/127) with no twin pregnancies. For the study population as a whole, the ongoing pregnancy rate was 42% (101/243) with 11% (11/101) twins. CONCLUSION: We conclude that the introduction of single embryo transfer in the first IVF/ICSI cycle is highly acceptable in women <38 years old.     

Delayed embryo transfer in an in-vitro fertilization programme: how to avoid working on Sunday.

It has previously been demonstrated that use of gonadotrophin releasing hormone analogues allows the administration of HCG to be delayed and makes it possible to avoid oocyte retrievals at weekends. In this study, we demonstrate that it is also possible to avoid embryo transfers on Sundays, by delaying transfers of Friday retrievals until Monday, without any apparent change in the results. This facility should improve the cost-effectiveness of assisted reproduction programmes.     

Fertilization and early embryology: The prognostic value of successful in-vitro fertilization in subsequent trials

A retrospective study comparing in-vitro fertilization (IVF)results in patients with previous successful trials versus newIVF patients was designed with the objective of evaluating theprognosis in both groups. Patients were divided into two groups:group A, 22 patients with previous conception in IVF trials;and group B, 235 new IVF patients. All patients had tubal factoras the only cause of infertility and were stimulated by a standardprotocol of gonadotrophin-releasing hormone agonist and humanmenopausal gonadotrophin. The pregnancy rate in group A (64%)was significantly higher than in group B (19%). In group A,77% of the patients had three or more grade 1 embryos per transferas compared with 16% in group B. In group A, 95.5% of patientshad easy embryo transfer as compared with 61.2% in group B.The results of the study demonstrated that patients with previousIVF pregnancies have a significantly higher pregnancy rate comparedwith the control group. The improved pregnancy rate is due tothe higher quality of embryos and the higher percentage of easyembryo transfers.     

Construction of an evidence-based integrated morphology cleavage embryo score for implantation potential of embryos scored and transferred on day 2 after oocyte retrieval

BACKGROUND: Evidence-based morphological embryo scoring models for ranking of implantation potential are still scarce, and the need for a precise model increases when aiming for singleton pregnancies. METHODS: Prospectively, 2266 IVF/ICSI double-embryo, day 2 transfers were studied. The five variables scored in 3- to 5-step scales for the embryos transferred are blastomere number (BL), fragmentation, blastomere size variation ('equality', EQ), symmetry of the cleavage and mononuclearity in the blastomeres (NU). The scoring results of embryos with an individual traceability from scoring to implantation, i.e. treatments resulting in either no implantation (n=1385) or twin implantation (n=228), were studied for prognostic potential. RESULTS: Although all five variables correlated highly with implantation potential, only BL, NU and EQ remained independently significant after regression analysis. The equation thus derived formed the basis for a 10-point integrated morphology cleavage (IMC) embryo score. A table with the scoring point for each possible combination of the embryo variables is presented. The scoring model was statistically validated on the singleton pregnancy group (n=653). CONCLUSIONS: We suggest that this IMC embryo scoring, incorporating cleavage stage and information on the variation in blastomere size and the number of mononucleated blastomeres, may optimize embryo ranking and selection for day 2 transfers.     

Frozen embryo transfers: implications of clinical and embryological factors on the pregnancy outcome

BACKGROUND: Frozen embryo transfers are characterized by impaired pregnancy outcome and increased incidence of pregnancy loss as compared with fresh IVF/ICSI embryo transfers. In this study, we performed a retrospective analysis of clinical and embryological factors that potentially influence the outcome of frozen embryo transfer. METHODS: We reviewed the outcome of 1242 frozen embryo transfers with respect to the age of the woman, the method of fertilization, embryo quality before and after freezing and the number of embryos transferred. RESULTS AND CONCLUSIONS: The pregnancy (positive hCG) and clinical pregnancy rates were 25.8 and 21.1%, respectively. A total of 107 (33.3%) of the 321 pregnancies identified by a positive hCG test miscarried either before (18.4%) or after (15%) the clinical recognition of gestational sac(s). The delivery rate for the frozen embryo transfers analysed was 17.2%. Our data revealed that the delivery rate after frozen embryo transfer was dependent on both the woman's age and the quality of embryos transferred, at the same time being unaffected by IVF/ICSI treatment. In addition, the increased woman's age at IVF/ICSI treatment was identified as the only parameter elevating the biochemical pregnancy rate, whereas the clinical abortion rate was found to be unrelated to the clinical or embryological parameters studied.