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.     

Embryonic soluble HLA-G as a marker of developmental potential in embryos

BACKGROUND: In human reproduction, embryo implantation is complex and poorly understood. At present, no single markers are used in routine treatment to assay biochemical functions of the human embryo. Soluble human leukocyte antigen-G (sHLA-G) could be considered a possible marker of embryo developmental potential. It is localized primarily on the extravillous trophoblast, making this antigen a potential mediator of immune interaction at the maternal-fetal interface during gestation. METHODS: Soluble-HLA-G levels were evaluated by an enzyme-linked immunosorbent assay (ELISA) employing monoclonal antibody MEM-G9. It was evaluated in 318 media of single embryo cultures. We correlated the presence of sHLA-G with embryo morphology and the pregnancy obtained in that treatment cycle. RESULTS: No correlation was found between embryo morphology and sHLA-G levels. Pregnancy was observed only when the medium of at least one transferred embryo contained sHLA-G. In 26 out of 66 patients, none of the obtained embryos showed any detectable sHLA-G molecules and no pregnancy occurred. CONCLUSIONS: From our results, we propose sHLA-G as a potential marker of embryo development: the sHLA-G ELISA can be a useful biochemical assay in addition to embryo morphology in embryo selection for transfer in IVF treatment if there are other embryos with the same morphology.     

A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos

The impact of cryopreservation on the implantation potential of early cleavage stage (day 2) embryos was assessed by analysing the outcome from > 5000 thawed embryos in relation to the outcome from a similar number of fresh embryos. Analysis of procedures in which all transferred embryos fulfilled equivalent defined criteria revealed no significant difference in the implantation rates (fetal hearts/100 embryos transferred) of fresh 4-cell embryos (16.6%) and fully intact thawed 4-cell embryos (16.9%). Although 2-cell embryos implanted at significantly lower rates, there was again no significant difference between fresh (6.5%) and fully intact thawed (7.2%) embryos. Similar analysis of all embryos (irrespective of cell number on day 2) demonstrated that the implantation potential of partially intact thawed embryos was related to the extent of blastomere loss with the implantation rate of embryos with 50% cell survival (5.4%) being approximately half the rate of fully intact embryos (11.3%). Combining the values obtained from 'pure' data for the implantation rates of embryos with defined levels of survival with their relative prevalence in the total population of thawed embryos gave a predicted number of implantations (441) which was similar to the observed outcome (463). This number was approximately 30% less than the number expected had the same embryos been transferred fresh (635). The results suggest that intact thawed embryos have the same implantation potential as equivalent fresh embryos and that the impact of cryopreservation is limited to blastomere loss which is directly related to loss of implantation potential. The observed frequency of blastomere loss results in a reduction of approximately 30% in the implantation potential of a population of embryos following cryopreservation.     

A prospective, randomized study comparing day 2 and day 3 embryo transfer in human IVF

It is believed that delayed transfer of embryos after IVF allows for a better selection of good quality embryos. Hence, the number of embryos and all other prognostic factors being equal, transfer of day 3 embryos should be associated with higher implantation and pregnancy rates than transfer of day 2 embryos. To investigate this hypothesis, a prospective randomized study was carried out to compare implantation and pregnancy rates between day 2 and day 3 transfers. The relationship between the embryo quality score of day 2 and day 3 embryos and their respective implantation rates was also analysed. In a 2 year period all patients undergoing infertility treatment and in whom at least seven normally fertilized oocytes were obtained were included in the study. A minimization procedure was performed taking into account the patient's age and the method of fertilization (IVF or intracytoplasmic sperm injection). By using a uniform policy of embryo transfer, the number of embryos transferred was similar in both groups. The outcome parameters were embryo quality, implantation and pregnancy rates. No difference was observed in implantation and pregnancy rates between transfers on day 2 versus day 3 (23.8 versus 23.8% and 47.9 versus 46.8% respectively). The incidence of embryos of moderate to poor quality was higher in embryos cultured for 3 days compared with those cultured for 2 days. It is concluded that the outcomes of embryo transfer in terms of implantation and pregnancy rates are comparable for day 2 and day 3 embryos, although the overall embryo quality score decreases when embryos are kept in culture till day 3.     

The implantation of every embryo facilitates the chances of the remaining embryos to implant in an IVF programme: a mathematical model to predict pregnancy and multiple pregnancy rates

BACKGROUND: We aimed to assess the validity of a theoretical mathematical model to predict the pregnancy rate and the multiple pregnancy rate in IVF/oocyte donation programmes on the basis of the implantation rate and the number of transferred embryos. METHODS: A total of 1835 embryo transfers corresponding to three different programmes in two centres with different implantation rates were analysed. Pregnancy and multiple pregnancy rates observed in the aforementioned programmes were compared with those obtained following different mathematical models. Four models were tested: binomial model, ground model, maternal variability model and collaborative model. The goodness of fit was performed by means of the maximum likelihood fit method. RESULTS: The binomial model could not predict the pregnancy rate, and especially the multiple pregnancy rate. The multiple pregnancy rate predicted following the binomial model was much lower than observed, up to 40-fold reduced. Ground model and maternal variability model adjusted to the data with more precision, but were still not accurate. Finally, the collaborative model reproduced with very great accuracy both pregnancy rate and the multiple pregnancy rate. A collaborative parameter of 22% was found, implying that the implantation probability of each embryo is increased by 22% for every embryo previously implanted. CONCLUSIONS: Embryonic implantation does not follow a binomial law, showing that the implantation is not independent from the number of embryos implanted. The best fit to the data is obtained following a collaborative model by which the implantation of one embryo is facilitated by the implantation of other embryo(s). The mathematical formula of the collaborative model predicts very accurately the pregnancy rate and the multiple pregnancy rate in IVF/oocyte donation programmes, based on the implantation rate of this specific programme and the number of embryos transferred up to five embryos. We recommend using the aforementioned formula to quantify the pregnancy rate and the risk of multiple pregnancy in the counselling of the infertile couple at embryo transfer. Such a formula is freely available at www.ifca.unican.es/matorras/mathpreg/.     

Computer-controlled, multilevel, morphometric analysis of blastomere size as biomarker of fragmentation and multinuclearity in human embryos

BACKGROUND: Little is known about blastomere size at different cleavage stages and its correlation with embryo quality in human embryos. Using a computer system for multilevel embryo morphology analysis we have analysed blastomeres of human embryos and correlated mean blastomere size with embryonic fragmentation and multinuclearity. METHODS: A consecutive cohort of 232 human 2-, 3- and 4-cell embryos from patients referred for ICSI treatment were included. Sequences of digital images were taken by focusing at 5- micro m intervals through the embryo. Blastomere sizes and number of nuclear structures were evaluated based on these sequences. The degree of embryonic fragmentation was evaluated by normal morphological assessment prior to transfer and correlated to the blastomere sizes. RESULTS: As a result of normal cell cleavage, mean blastomere size decreased significantly from a volume of 0.28 x 10(6) microm(3) at the 2-cell stage to 0.15 x 10(6) microm(3) at the 4-cell stage (P < 0.001). Mean blastomere size decreased significantly (P < 0.001) with increasing degree of embryonic fragmentation, where highly fragmented embryos showed a 43-67% reduction in blastomere volume compared with embryos with no fragmentation. Multinucleated blastomeres were significantly larger than non-multinucleated blastomeres (P < 0.001). On average, multinucleated blastomeres were 51.5, 67.8 and 73.1% larger than their non-multinucleated sibling blastomeres at the 2-, 3- and 4-cell stage, respectively. Furthermore, the average volume of non-multinucleated blastomeres originating from multinucleated embryos was significantly smaller than the average volume of the blastomeres from mononucleated embryos (P < 0.001). CONCLUSIONS: The results of this study show that the average blastomere size is significantly affected by degree of fragmentation and multinuclearity, and that computer-assisted, multilevel analysis of blastomere size may function as a biomarker for embryo quality.     

Enhancement of embryo developmental potential by a single administration of GnRH agonist at the time of implantation

BACKGROUND: Several reports have shown that inadvertent administration of a GnRH agonist in the luteal phase does not compromise pregnancy. Moreover, some studies suggested that, unexpectedly, the embryo developmental potential is improved in these conditions. This prospective controlled study was designed to test this hypothesis. METHODS: In an oocyte donation programme, oocytes from each donor (n = 138) were shared by two recipients, one of whom was given a single dose of a GnRH agonist (0.1 mg triptorelin) 6 days after ICSI, and the other received placebo at the same time. RESULTS: Oocyte recipients treated with GnRH agonist 6 days after ICSI had higher implantation (36.9 versus 25.1%), twin pregnancy (16.7 versus 3.6%), twin delivery (13.8 versus 2.2%) and birth (31.1 versus 21.5%) rates and similar miscarriage and abortion rates as compared with the placebo group. CONCLUSIONS: GnRH agonist administration at the time of implantation enhances embryo developmental potential, probably by a direct effect on the embryo.     

Morphological evaluation of human embryos and derivation of an embryo quality scoring system specific for day 3 embryos: a preliminary study

A scoring system specific for day 3 embryos has not been extensively explored. Most IVF laboratories continue to grade embryos solely on the basis of cell number and percentage fragmentation as was traditionally done for day 2 embryos. Additional morphological features, some unique to day 3 embryos, may be useful in selecting embryos most likely to blastulate and implant. The objective of this study was to derive an embryo scoring system for day 3 transfers which is predictive of positive pregnancy outcomes. A total of 316 transferred embryos from 93 patients was recorded on videotape and evaluated. The following parameters were used to grade the embryos: cell number, fragmentation pattern (FP), cytoplasmic pitting, compaction, equal sized blastomeres, blastomere expansion and absence of vacuoles. The clinical pregnancy rate was 41.9%, with an implantation rate of 18% per embryo transferred. The mean number of embryos transferred per patient was 3.4. Three formulae were derived to score embryo quality in each transfer based on the average score of individual embryos transferred. In the first scoring system, cell number alone was used to predict pregnancy outcome. The second scoring system was based on blastomere number and the observed FP. The third scoring system utilized both blastomere number and FP but also combined this with five morphological criteria to yield a final day 3 embryo quality (D3EQ) score. We found the D3EQ score to be prognostic of pregnancy outcome. This study suggests that although cell number and FP are certainly predictors of positive pregnancy outcomes, additional parameters specific to day 3 embryos should be used to stratify a cohort of embryos further.     

Influence of early ICSI-derived embryo sHLA-G expression on pregnancy and implantation rates: a prospective study

BACKGROUND: We have previously reported the retrospective observation that when at least one embryo, transferred on day 3, expressed sHLA-G above the geometric mean (sHLA-G+) 46 h post-ICSI, there was a marked improvement in both pregnancy (PR) and implantation (IR) rates. METHODS: The media surrounding individual embryos derived from ICSI performed on oocytes from 482 women < or =43 years of age were tested for sHLA-G expression by specific ELISA. RESULTS: We report here prospective results showing improved IVF results following the transfer of 'good quality' embryos (7-9 cells with <20% fragmentation) by preferentially including at least one sHLA-G+ embryos. PR and IR for women < or =38 years were 63% and 32% when one transferred embryo was sHLA-G+, and 69% and 36% when at least two embryos were sHLA-G+. When none of the embryos transferred was sHLA-G+, PR and IR were 25% and 13%, respectively. Comparable PR and IR for women 39-43 years were 29% and 11% when none of the transferred embryos were sHLA-G+; 38% and 15% when at least one sHLA-G+ embryo was transferred; and 61% and 26% when at least two 2 sHLA-G+ embryos were transferred. The data were stratified by patient age. CONCLUSIONS: PR and IR increased with the addition of each sHLA-G+ embryo, regardless of age. While there are significant barriers to routine embryo sHLA-G testing, we believe that if implemented, this would provide a mechanism for optimizing IVF PR while minimizing the risk of multiple pregnancies.     

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.     

Laser-assisted hatching increases pregnancy and implantation rates in cryopreserved embryos that were allowed to cleave in vitro after thawing: a prospective randomized study

BACKGROUND: Cryopreservation of embryos may lead to zona hardening that may compromise in vivo hatching and implantation following thawing and transfer. Assisted hatching (AH) has been advocated as a means of assisting the natural hatching process and enhancing implantation. METHODS: The aim of this study was to assess in a prospective randomized manner the effect of laser-assisted hatching (LAH) on implantation as well as clinical and multiple pregnancy rates (the primary outcome) after the transfer of frozen-thawed embryos. All embryos were thawed the day before transfer, and LAH was performed the next day on embryos that cleaved. Control group consisted of embryos that were transferred without AH. RESULTS: The performance of LAH significantly increased implantation (9.9 versus 20.1%, P < 0.01), clinical pregnancy (27.3 versus 40.9, P < 0.05) and multiple pregnancy rates (16 versus 40.3%, P < 0.07). In the LAH group, significantly more excess embryos that were left in culture hatched in vitro. CONCLUSIONS: LAH improves the outcome of frozen-thawed embryo transfer when performed before transfer on embryos that were allowed to cleave.     

Parameters guiding selection of best embryos for transfer after cryopreservation: a reappraisal

BACKGROUND: The purpose of this study was to evaluate the respective influences of blastomere survival and resumption of mitosis on the outcome of frozen-thawed embryos. METHODS: A retrospective analysis was performed in our centre on 363 thawing cycles, involving 4-cell day 2 grade 1 embryos with <10% fragmentation. RESULTS: A higher implantation rate per transferred embryo was observed when all transferred embryos were characterized by fully intact blastomeres (100% blastomere survival) as compared with damaged embryos (50 or 75% blastomere survival) (22.0 versus 7.2%; P < 0.0001). Moreover, the implantation rate per transferred embryo was significantly higher for cleaved embryos compared with uncleaved embryos (19.7 versus 3%; P < 0.0001). Transfer of fully intact, cleaved embryos resulted in the highest implantation rates compared with transfer of damaged and uncleaved embryos (27.4 versus 0%; P < 0.0001). Intermediate implantation rates were observed when only one of the two criteria was fulfilled (13 versus 11% respectively; P > 0.05). Multivariate analysis showed that the clinical pregnancy rate was influenced by both criteria (odds ratio = 3.4 for transfer of embryos with six or more cells versus embryos with less than six cells. CONCLUSION: The results of our study suggest that the most important factor to predict further embryo development is the total number of blastomeres in transferred embryos, however they are obtained (good survival and/or resumption of mitosis).     

Pregnancy: Effect of pentoxifylline on implantation and post-implantation development of mouse embryos in vitro

Previous work from our laboratory has revealed that brief exposureof mouse zygotes to 3.6 and 7.2 mM pentoxifylline reduced cellnumbers in mouse embryos reaching the blastocyst stage in vitro.Since a decrease in cell numbers may impair further development,the present study aimed to assess the implantation in vitroand the development beyond implantation of these embryos. Blastocystscultured from the one-cell stage in the presence of 5, 10 and50 µM pentoxifylline, i.e. concentrations expected inthe secretions of the genital tract after oral administration,were shown to develop normally to the egg cylinder stage invitro. Blastocysts, obtained after culture in vitro of zygotesexposed 29 h after human chorionic gonadotrophin (HCG) for 30min to 3.6 and 7.2 mM pentoxifylline, i.e. the concentrationsused for enhancing sperm function in vitro, could implant buttheir development to the egg cylinder stage was found to beimpaired. The same effect was noticed if blastocysts, grownin vitro from the one-cell stage, were exposed to 3.6 and 7.2mM pentoxifylline for 30 min in vitro 120 h after HCG. Thesefindings imply that when pentoxifylline is used to enhance spermfunction in vitro, it should be washed out of the preparationused for insemination.     

Live birth rate is significantly higher after blastocyst transfer than after cleavage-stage embryo transfer when at least four embryos are available on day 3 of embryo culture. A randomized prospective study

INTRODUCTION: In a randomized controlled trial, we assessed whether pregnancy outcome would be improved by extending embryo culture to day 5 and transferring a blastocyst in patients with at least four good-quality embryos on day 3. METHODS: Multifollicular ovarian stimulation was performed with a GnRH agonist in 44% of patients and with a GnRH antagonist in 56%. Overall, 164 patients younger than 37 years fulfilled embryo quality criteria (at least four having at least six cells on the morning of day 3, maximum 20% anucleate fragments) on the third day of culture and were randomized to the day 3 (n = 84) or day 5 (n = 80) groups. Equal numbers of embryos (n = 2) were transferred in each group. RESULTS: Demographics, stimulation parameters and embryological data were comparable in the two groups. Blastocyst-stage transfer resulted in a significantly higher ongoing pregnancy rate [51.3 versus 27.4%; odds ratio (OR) 2.78, 95% confidence interval (CI) 1.45-5.34] and live birth rate (47.5 versus 27.4%; OR 2.40, 95% CI 1.25-4.59) compared with day-3 embryo transfer. A high twin birth rate was observed in both groups (36.8 versus 30.4%; P > 0.05). CONCLUSIONS: A threshold of four good embryos on the third day of embryo culture appears to indicate that the patient will benefit from embryo transfer at the blastocyst stage and have a better chance of achieving a live delivery than with cleavage-stage embryo transfer.     

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.     

One year's experience with elective transfer of two good quality embryos in the human in-vitro fertilization and intracytoplasmic sperm injection programmes

High incidences of multiple pregnancies, after transferringa maximum of three embryos, were observed after in-vitro fertilization(IVF) treatment. In a randomized study, it was demonstratedthat, after taking into account embryo quality and other positivelyinterfering parameters, an elective transfer of two good qualityembryos does not significantly influence the pregnancy rate.The intracytoplasmic sperm injection (ICSI) technique was successfullydeveloped in the meantime and high incidences of multiple pregnancieswere also obtained after ICSI. The question arose whether afterICSI there was also room for elective double embryo transferin a well-defined patient group. This report covers 1 year of IVF and ICSI treatment and theresults are presented in relation to the number of embryos transferred.The embryo development is similar for zygotes obtained afterIVF and ICSI; for both techniques 63% of the zygotes developto type A-B embryos and 13% to type C embryos. There is alsono difference in the pregnancy rate after ICSI or IVF. Globally,after IVF, 307 out of the 766 double and triple transfers (40.1%)and 317 out of 774 double and triple transfers (40.9%) afterICSI resulted in a positive HCG. After IVF, 73.9% (227) andafter ICSI 76.3% (242) of the pregnancies were evolutive. Neitherwas there any difference between the two techniques as regardsthe implantation rate per transferred embryo. After IVF, 22.8%of the transferred embryos implanted compared with 21.8% afterICSI. When the elective double embryo transfers were compared,no difference was found between IVF and ICSI. After IVF, 102of the 211 elective double transfers (48.1%) resulted in a pregnancyversus 93 out of 225 (41.3%) after ICSI [not significant (NS)].A high implantation rate per transferred embryo (IVF: 33.2%;ICSI: 26.9%, NS) was obtained in this elective double transfercategory, as was also reported in the randomized study. Thesedata confirm the results obtained in our randomized study andthe effectiveness of the elective double embryo transfer forIVF as well as for ICSI.     

Influence of position and length of uterus on implantation and clinical pregnancy rates in IVF and embryo transfer treatment cycles

In a prospective study of 807 consecutive women shown to have an apparently normal uterus after hysterosalpingography, hysteroscopy or pelvic ultrasonography prior to IVF or intracytoplasmic sperm injection (ICSI) and embryo transfer, the position and length of the uterine cavity was measured routinely at a pre-treatment mock transfer procedure. The apparent length of the uterine cavity was <7 cm in 128 women (group 1), 7-9 cm in 594 women (group 2) and >9 cm in 85 women (group 3). The uterus was noted to be retroverted in 38. 2% (308) women. The embryo transfer catheter was advanced to 5 mm from the uterine fundus based on the previously determined cavity length in all the embryo transfer procedures at 48 h after oocyte collection. Implantation and clinical pregnancy rates were not significantly different with respect to position of the uterus, difficulties encountered in passage of the catheter, mean age of the women, aetiology or duration of infertility or embryology events. An apparently greater cavity length was seen in older and/or parous women, but the difference was not statistically significant. Although the highest implantation and clinical pregnancy rates were seen in women with a cavity length of 7-9 cm (group 2) the differences were not statistically significant: group 1, 18.9 and 36. 7%; group 2, 21.0 and 46.5%; and group 3, 17.3 and 32.9% respectively. The incidence of ectopic pregnancy per reported clinical pregnancy was highest in group 1 women, being 14.9% (7/47) in comparison with group 2 (1.8%, 5/276) and group 3 (0%, 0/27) (P: < 0.0005), suggesting that the size of the uterus is a critical factor in the aetiology of ectopic pregnancy in IVF/ICSI-embryo transfer.     

Effect of retinoic acid on implantation and post-implantation development of mouse embryos in vitro

BACKGROUND: This study was designed to examine the embryotoxic potential of retinoic acid (RA) at the blastocyst stage and during early post-implantation development of mouse embryos in vitro. METHODS AND RESULTS: All-trans retinoic acid (t-RA) was administered to ICR mice embryos at a dose level of 0, 0.001 micromol/l, 0.1 micromol/l and 10 micromol/l throughout in-vitro culture. A total of 404 embryos was randomly assigned to all different dose groups. The percentage of embryos in later stages of development changed depending upon the dose of RA used. Exposure to 10 micromol/l of t-RA at the blastocyst stage, implanted blastocyst stage or early oocyte stage was also found to cause different degrees of retardation of embryo development and embryo death. These findings demonstrate, for the first time, that RA exerts an adverse effect on embryo growth during the early post-implantation stages of development, in comparison with day 3 to day 8 of gestation in vivo. CONCLUSIONS: Although isotretinoin (13-cis-retinoic acid) is effective for the therapy of cystic acne and other dermatological disorders, retinoid treatment should be avoided at the early post-implantation stage of gestation.     

Luteal phase ovarian oestrogen is not essential for implantation and maintenance of pregnancy from surrogate embryo transfer in the rhesus monkey

The aim of this study was to investigate whether luteal phaseovarian oestrogen is required for blastocyst implantation andpregnancy maintenance in the rhesus monkey. Preimplantationembryos were retrieved from naturally ovulated, mated embryodonor monkeys. In group I, developmentally normal, age- andstage-matched embryos were transferred to recipient monkeysshowing naturally synchronized ovulatory cycles. Immediatelyprior to embryo transfer, recipients were subjected to bilateralovariectomy, and following transfer they were treated with i.m.injections of either progesterone (group Ia, n= 4), or oestradiol+ progesterone (group Ib, n= 2). Recipient monkeys of groupIc (n= 4) were subjected to sham ovariectomy and vehicle injection.In group Ia, progesterone supplementation alone led to threepregnancies and live births. In group Ib, there was one livebirth. In the control group Ic, four transfers resulted in twolive births and one abortion on cycle day 58. Analysis of serumprogesterone and oestradiol profiles showed that oestradiolhad declined to undetectable levels within 3–5 days afterovariectomy in group Ia recipients, and the area under the curveof serum oestrogen concentrations during the peri-implantationperiod (days 10–20 after ovulation) were less (p< 0.001)in group Ia compared with group Ic. There were no changes inthe area under the curve among serum progesterone concentrationsin all the subgroups. In group II, long-term ovariectomizedembryo recipients (n= 4) were primed with oestradiol till cycleday 11 of simulated transfer cycle, and received progesteronetreatment from cycle day 10 till the end of the experiment.Of four transfers, live births were recorded in two cases, whilein one case abortion occurred on cycle day 66. Serum oestradiolconcentrations were undetectable during the presumptive peri-implantationperiod of pregnancy cycles in group II recipient monkeys. Noyes‘dating of endometrial samples collected from both groups ondays 5–7 after the oestrogen rise revealed that endometrialhistology synchronized well with those found during days 3–5after ovulation in normal menstrual cycle. We conclude thatluteal phase ovarian oestrogen is not essential for progesterone-dependentendometrial receptivity and response leading to implantationand pregnancy maintenance in the rhesus monkey.     

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.