Embryo Implantation Potential Research Articles

Secretion of soluble HLA-G by day 3 human embryos associated with higher pregnancy and implantation rates: assay of culture media using a new ELISA kit

Identification of chemical markers in human embryo culture media that relate to embryo quality and implantation potential could be an invaluable tool in embryo selection for transfer. Embryonic secretion of soluble human leukocyte antigen G (sHLA-G) has been postulated to be a marker for ‘embryonic competence'. This study examines sHLA-G concentrations in day 3 culture media droplets of embryos that were selected for transfer, as well as those being cryopreserved. A total of 712 embryo culture supernatants from 83 patients were assayed. Soluble HLA-G was detected in 306 of the 712 samples tested. In the 58 transfers in which at least one embryo selected for embryo transfer was positive for sHLA-G, the pregnancy rate was 64% (37/58) and the implantation rate per embryo transferred was 38%. In contrast, patients receiving only embryos that were negative for sHLA-G had both a lower pregnancy rate of 36% (9/25; P < 0.05) and a decreased implantation rate (19%; P < 0.05). Expression of sHLA-G was also related to increasing cell stage. Concentration of sHLA-G in embryo culture media was variable and in the low range (3–10 ng/ml). These data suggest an association between implantation potential and embryonic secretion of sHLA-G. The commercial assay kit utilized allowed for same day assessment of sHLA-G secretion. Addition of sHLA-G status to traditional morphological criteria may be useful as a clinical tool for embryo selection.

Does 'soluble' HLA-G really exist? Another twist to the tale

HLA-G is thought to play a key role in implantation by modulating cytokine secretion to control trophoblast invasion and to maintain a local immunosuppressive state. It differs from other class I molecules in that the gene can be alternatively spliced to produce four membrane-bound (G1, G2, G3 and G4) and three soluble isoforms (G5, G6 and G7). The soluble isoforms have recently attracted attention as their levels may be diagnostic of poor trophoblast invasion in miscarriage or pre-eclampsia and the implantation potential of IVF embryos. Although the expression and function of HLA-G2, G3, G4 and G7 has previously been a matter of debate, until now it has been generally accepted that soluble HLA-G5 and HLA-G6 are both expressed and secreted by trophoblast. However, Blaschitz et al. (2005) have reinvestigated this question and come to the surprising conclusion that they are not. They have shown that trophoblast only expresses the membrane-bound HLA-G1 isoform and not soluble HLA-G5 and -G6. Furthermore, although soluble HLA-G could be found in trophoblast culture supernatants, it appears not to be derived by alternative splicing but by the cleavage of HLA-G1. The source of the soluble HLA-G may not matter from a diagnostic perspective, but these findings, if confirmed, have important implications for our understanding of the biology of HLA-G.

Effect of coasting on the implantation potential of embryos transferred after cryopreservation and thawing

To examine the effect of withholding gonadotropins on the outcome of embryos after cryopreservation and thawing. Retrospective clinical evaluation of patients having cryopreserved-thawed ET trials with coasting during the corresponding ovarian stimulation cycle. Academic tertiary clinical care unit. Patients with cryopreserved embryos having coasting in their fresh IVF cycle and age-matched controls without coasting, both groups receiving the same stimulation protocol (long GnRH agonist plus recombinant FSH). All patients had a cycle in which embryos were transferred fresh and a cycle of thawing of cryopreserved embryos with the aim of transferring in a steroid-supplemented cycle. Embryo survival, implantation, and clinical pregnancy rates. Post-thawing embryo survival (66.4% vs. 73%), implantation (12.3% vs. 13.0%), and clinical pregnancy rates (31.5% vs. 38.0%) were similar in study and control groups, respectively. Patients with coasting for > or =3 days had significantly lower post-thawing embryo survival rates compared with patients having shorter duration of coasting (<3 days) and controls. Implantation and pregnancy rates, however, were similar in the three groups. Coasting did not seem to have a detrimental effect on oocyte and embryo quality because the implantation competence of transferred concept after cryopreservation and thawing was similar to that of controls. However, prolonged coasting (> or =3 days) had a subtle negative impact on the post-thaw survival rate.

The Incidence of Cohort Multinucleation Does Not Impact Implantation of Sibling Mononucleated Embryos

The presence of multinucleation in an embryo has been shown to be indicative of chromosomal abnormalities. Due to the complex relationship between sibling embryos in a group-cultured cohort, it is unclear if a higher incidence of multinucleation results in a lower implantation potential of sibling mononucleated embryos. High rates of cohort multinucleation have previously been correlated with various factors relating to aggressive ovarian stimulation, rapid ovarian stimulation response and a greater number of oocytes retrieved.

Assessment of Soluble HLA-G Levels in Day 3 Human Embryo Culture Media Using a New ELISA Kit: Is There a Relationship to Embryo Quality, Development and Implantation Potential?

Identification of chemical markers in human embryo culture media that correlate to embryo quality and implantation potential could be an invaluable tool in embryo selection for transfer. Several studies have suggested that high quality embryos secrete soluble HLA-G . This study examines sHLA-G levels in both embryos that are selected for transfer as well as those kept in culture for cryopreservation at the blasocyst stage.

Early Embryo Cleavage and Mononucleate Blastomeres as Predictors of Embryo Implantation Potential

To evaluate the predictive value of early embryo cleavage (25 - 27 hours post insemination/ICSI) of day two 4-cell preembryos which showed a single nucleus in each blastomere (“4-cell / 4 nuclei” preembryo) at the time of transfer. In previous studies, we have demonstrated that transfer of day two 4-cell preembryos with a synchronous appearance of nucleation yields a significantly higher implantation rate compared to transfer of 4-cell preembryos with an asynchronous appearance of nucleation (0 - 3 blastomeres showing a single nucleus). Also early embryo cleavage has been claimed to be an important marker for implantation potential of an embryo. Retrospective study. Single embryo transfers. Early cleaving preembryos, which were “4-cell / 4 nuclei” preembryos at the time of transfer, were primarily chosen for transfer. If no such embryo was available, a “4-cell / 4 nuclei” preembryo which had not cleaved early was taken for transfer. 364 consecutive fresh day two single embryo transfers of “4-cell / 4 nuclei” preembryos were performed from February 2004 to February 2005. Implantation rates (at least a gestational sac at ultrasound examination at 7 weeks of gestation) of “4-cell / 4 nuclei” preembryos which had or had not cleaved early were analysed. The pregnancy/implantation rates after transfer of day two “4-cell / 4 nuclei” preembryos with or without early cleavage are shown in the table. Tabled 1 The observation early embryo cleavage adds no extra in predicting implantation potential of a day two 4-cell preembryo with mononucleate blastomeres.

Impact of assisted hatching on ART outcome in women with endometriosis

Assisted hatching can improve the implantation rate in cycles with poor outcome. The impact of assisted hatching in embryos from women with endometriosis is not known. Therefore, the hypothesis that the implantation potential of embryos obtained from women with endometriosis can be improved with assisted hatching was tested. In a prospective randomized study, transfer embryos obtained from 60 women with endometriosis were hatched using a laser system and compared to embryos obtained from patients with the same diagnosis which were left intact (n = 30). The characteristics of cycles were similar between groups. The pregnancy (40% zona intact, 28.3% assisted hatching), and implantation rates (19.4% zona intact, 17.8% assisted hatching) did not differ in endometriosis cycles regardless of assisted hatching. Assisted hatching does not improve outcome in women with endometriosis undergoing assisted reproduction.

Expression of genes regulating chromosome segregation, the cell cycle and apoptosis during human preimplantation development

Appropriate gene expression is vital for the regulation of developmental processes. Despite this fact there is a remarkable paucity of information concerning gene activity during preimplantation development. We employed reverse transcription and real-time fluorescent PCR to quantify the expression of nine genes (BRCA1, BRCA2, ATM, TP53, RB1, MAD2, BUB1, APC and beta-actin) in oocytes and embryos. A full characterization of all genes was achieved in 42 embryos and four oocytes. The genes analysed have a variety of important cellular functions. Oocytes displayed relatively high levels of mRNA transcripts, while 2-3-cell embryos were seen to contain very little mRNA from any of the genes examined. Recovery of expression levels was not seen until the 4-cell stage or later, with the presumptive activation of the embryonic genome. Some genes displayed sharp increases in expression in embryos composed of 4-8 cells, but, for most, maximum expression was not achieved until the blastocyst stage. Our data show that it is possible to define characteristic gene expression profiles for each stage of human preimplantation development. The identification of genes active at defined preimplantation phases may provide clues to the cellular pathways utilized at specific stages of development. Expression of genes that function in DNA repair pathways indicate that DNA damage may be common at the cleavage stage. We suggest that specific patterns of gene expression may be indicative of embryo implantation potential.

Zona pellucida thickness and clinical pregnancy outcome following in vitro fertilization

Objective To investigate possible correlations of ZPT or ZPTV with clinical outcome following in vitro fertilization. Methods Two hundred forty-six embryos were selected for transfer on day 3 with clear image record from 81 IVF–ET cycles. The laser system measurement software was used to measure the ZTP value of each embryo and the ZPTV was computed. Statistical analysis was done using the ANOVA or Chi-square test. Results Both ZPT and ZPTV declined with women's age. The mean ZPTV of embryos from patients between 30 and 34 years old was significantly higher than that from patients older than 35 ( P < 0.001). The ZPTV in pregnancy group was significantly higher than that in nonpregnancy group( P < 0.005). The clinical pregnancy rate in the group with ZPTV more than 20% was significantly higher than that in the group with ZPTV less than 20% ( P < 0.05). The ZPTV of high grade embryos was significantly higher than that of low grade embryos ( P < 0.001). Conclusions ZPTV rather than ZPT is an effective parameter to evaluate the embryo quality. Increasing the ZPTV may enhance embryo implantation potential.

Support for the contention that sperm with abnormal sperm chromatin structure assays are associated with reduced embryo implantation potential

Support for the contention that sperm with abnormal sperm chromatin structure assays are associated with reduced embryo implantation potential

Impact of the presence of one or more multinucleated blastomeres on the developmental potential of the embryo to the blastocyst stage

A retrospective study of 5,982 embryos in 619 blastocyst-stage embryo transfer cycles revealed that detection of multinucleated blastomeres either on day 2 or 3 signifies a poor prognosis for blastocyst formation and that no good-quality blastocyst can be expected from an embryo with more than one multinucleated blastomere. Patients should be counseled regarding a poor prognosis when multiple embryos with multinucleated blastomeres are present.

Freeze–thaw programmes rescue the implantation of day 6 blastocysts

The developmental rate of a blastocyst is considered one of the main estimates for evaluating the implantation potential of embryos. Day 6 blastocysts have been reported to be much less viable than day 5 blastocysts. Regarding implantation, the implantation window is advanced due to a background of high sex hormones, and slower growing embryos may not implant because of possible desynchrony with the implantation window. The aim of this study was to investigate the efficacy of cryopreservation of such embryos and subsequent synchronization of embryo transfer with endometrial status. The results of 122 day 6 blastocysts transferred in the clinic were retrospectively examined. Pregnancy rates were compared between the stimulation cycle and hormone replacement cycle in terms of the method of endometrial preparation. Fifty-five day 6 blastocysts were transferred onto the stimulation cycle endometrium in 37 women, resulting in a 5.5% viable pregnancy rate. On the other hand, 67 day 6 blastocysts were transferred onto endometrium prepared by exogenous hormones in 40 women, resulting in a 26.9% viable pregnancy rate ( P < 0.01). Consequently, the difference was highly significant. In conclusion, synchronous transfer of slow-growing embryos using the freeze–thaw technique contributes to a positive outcome.

Single point determination of day 2 embryos yield comparable implantation rate when compared to sequential assessment

Single point determination of day 2 embryos yield comparable implantation rate when compared to sequential assessment

Predictive value of embryo grading for embryos with known outcomes

Predictive value of embryo grading for embryos with known outcomes

Maturation, embryo development and clinical outcomes in ICSI treatment cycles with completely immature oocytes at the removal of cumulus cells

It has been reported that in-vitro matured oocytes have lower developmental potential than in-vivo matured oocytes. Usually embryos derived from in-vitro matured oocytes are de-prioritized for transfer. Occasionally combined transfer of embryos derived from in-vitro and in-vivo oocytes is performed if there are not enough embryos from in-vivo matured oocytes for an appropriate ET. It is hard to tell if embryos derived from in-vitro matured oocytes attribute to pregnancies or live births under this situation. Few data is available about the full term development of these embryos that are derived from superovulated immature oocytes. In this study we retrospectively analyzed the clinical outcome of ICSI treatment cycles containing only embryos derived from in-vitro matured metaphase II (MII) and metaphase I (MI) oocytes. Retrospective chart review from January 2001 to March 2004. All the cycles included in this study contained no mature oocytes at the removal of cumulus cells. A total of 139 immature oocytes from 38 ICSI treatment cycles were included in the study. Among them 94 were at MI and the other 45 at germinal vesicle (GV) stage. These oocytes were cultured in 5% O2, 5% CO2 and 90% N2 incubator for 4–6 hours prior to injection. Oocyte maturation was checked just prior to ICSI and recorded. All MI oocytes were subject to ICSI along with vitro-matured MII oocytes. Fertilization, embryo development and clinical outcome were recorded. Embryo transfer was performed in the morning of day 3. Remaining embryos after transfer were cultured to blastocyst for potential freezing as is standard in our practice. Among the 38 cases, 29, 6 and 3 cases fell within the range of 1–4 , 5–8, >8 oocytes retrieved respectively according to oocyte number. After 4–6 hours culture in vitro, 65 MII matured from 94 MI (69.1%) and 15 MI progressed from 45GV oocytes (33.3%). ICSI was not performed in 2 cycles because all oocytes remained at GV. In total, 109 MII/MI matured oocytes were injected. Fifty-one (47%) fertilized with 2PN and cleaved. The proportion of good quality embryos (8-cell stage with < or =20% fragmentation on day 3) was 37.3% (19/51). There was no embryo transfer in 16 ICSI cycles due to absolute fertilization failure. Thirty-eight embryos were transferred back to the uterus in the other 20 cycles, resulting in 2 singleton pregnancies. The implantation rate was 5.3%. Unfortunately both pregnancies resulted in spontaneous abortions. Six blastocysts were frozen on day 5 from 2 cycles. No pregnancy was obtained from one thawing tranfer cycle with 2 frozen blastocysts. Four frozen blastocysts remain in storage for later thawing. Although this is a small study, it can be concluded that embryo implantation and further developmental potential is heavily compromised after ICSI of superovulated, in vitro matured occytes. This limitation is most likely reflects an underlying patient or ovarian issue rather than with in vitro maturation itself. Patients should therefore be advised of the potential implications when this situation occurs.

Soluble HLA-G measurement in the medium surrounding individual day 2 embryos in culture: A strong predictor of embryo implantation and viable pregnancy potential

We examined whether the expression of sHLA-G, a non-classical class I major histocompatibility complex molecule, in the culture media day 2 embryos was predictive of viable implantation (defined as an ultrasound-confirmed pregnancy with heart-beat) post “fresh” embryo transfer.

Aromatase activity in granulosa cells as a predictor of pregnancy potential

The ability to predict the potential of an oocyte to sustain embryo development in vitro would be an advantageous tool for reproductive technology. Currently, the only measure of oocyte quality is based on cumulus expansion or nuclear maturation. Neither of these observations predicts the degree of cytoplasmic maturation, which is critical for normal embryo development. The unique cell-to-cell interaction between the oocyte and surrounding granulosa cells (GCs) provides an opportunity to measure oocyte competence by quantifying properties of the supporting GCs. We have observed that the capability of GCs to synthesize estrogen, as measured by aromatase activity (AA), is related to pregnancy success of patients being treated for infertility. Evaluation of AA may provide a reliable non-invasive method of predicting implantation potential of embryos.

Improved Outcome of Frozen-Thawed Blastocyst Transfer with Menezo's two-step Thawing Compared to the Stepwise Thawing Protocol

To compare the effects of two different blastocyst thawing protocols on implantation, pregnancy, and live birth rates. Ninety four consecutive frozen-thawed blastocyst transfers from 1996 to 2002 were retrospectively analyzed. Blastocysts were cryopreserved using Menezo's two-step slow freezing protocol. Frozen blastocysts were thawed for transfer according to Menezo's stepwise or two-step protocol. Immediately after thawing, assisted hatching was performed and all embryos were cultured for 3 to 5 h before transfer. Only viable embryos were selected for transfer. Implantation, pregnancy (determined by the presence of fetal cardiac activity), and live birth rates were significantly higher with two-step (25, 45.7, and 42.9%) than with stepwise thawing protocol (9.2,18.6, and 16.9%, P < 0.01). The percentage of gestational sacs resulting in live babies was higher with two-step thawing (76.6%) than with stepwise thawing (50%, P = 0.04). We conclude that the two-step thawing protocol offers advantages over the stepwise method. The two-step thawing protocol dramatically increased embryo implantation potential, resulting in higher pregnancy rate, and subsequent live birth rate, after frozen blastocyst transfer.

Developmental potential of human pronuclear zygotes in relation to their pronuclear orientation.

Pronuclear zygote morphology has gained much attention recently due to its possible value in predicting implantation and pregnancy. The present study was performed to assess the developmental potential of zygotes with four different pronuclear orientations. This prospective study involved 150 IVF and 190 ICSI patients seeking fertility treatment. Pronuclear zygotes were classified for orientation of the pronuclei in relation to the second polar body placed at the 6 o'clock position. Four types of pronuclear (PN) zygote were recognized, namely PN(1), PN(2), PN(3) and PN(4). The main outcome measures were early cleavage rate, quality of embryos, and implantation and pregnancy rates. The most common types of pronuclei orientations were PN(1) (31.5%) and PN(2) (29.3%), followed by PN(3) (20.5%) and PN(4) (18.5%). A significantly higher proportion of zygotes with PN(1) and PN(4) types of pronuclear orientation underwent early cleavage and developed into grade I embryos compared with other types (P < 0.0001). There was a tendency for higher implantation and pregnancy rates among patients who received embryos developed from PN(1)- and PN(4)-type oocytes, but this was not statistically significant. Zygotes exhibit four types of pronuclear orientation, and this is independent of the fertilizing spermatozoon or its entry point into the oocytes, whether IVF or ICSI is employed. Early cleavage was associated with PN(1)- and PN(4)-type zygotes, but implantation and pregnancy rates were not associated with pronuclear orientation. Implantation and pregnancy rates tended to be higher for embryos developed from PN(1) and PN(4) pronuclear zygotes. Further studies on a combination of pronuclear orientation and equality of nucleoli may provide a better guide to the implantation potential of embryos.

Transfer of nonselected transferable day 3 embryos in low embryo producers

Objective To examine the implantation potential of embryos from assisted reproductive technology cycles with low embryo production and to assess the effects of clinical variables and embryo scores (ES) on pregnancy outcome. Design Prospective clinical study. Setting Assisted reproductive technology unit in a tertiary medical center. Patient(s) From July 1998 to December 2001, 280 cycles in 229 infertile couples produced a limited number of one, two, or three embryos 3 days after oocyte retrieval and underwent fresh embryo transfer (ET). Intervention(s) Embryos with two or more blastomeres were scored and transferred. Main outcome measure(s) ES and implantation rate per ET. Result(s) Of 863 fresh ET cycles during the study period, 32.4% (280) were low embryo producers. Among them, there were no significant differences in average ES of individual embryos in single, dual, or triple ET or in embryos obtained from patients with low or high E 2 responses, or young or old age. Embryos derived from conventional IVF had a better ES than those derived from intracytoplasmic sperm injection. The clinical pregnancy rate was strongly correlated with the cumulative ES. Implantation rates were similar among and between groups, with an average rate of 15.9%. Conclusion(s) Embryos of low embryo producers had an inherently low implantation potential that appeared to be unrelated to the number of embryos transferred, female age, ovarian E 2 genesis, or fertilization method. The cumulative ES can serve as a predictor of pregnancy.