Embryo Implantation Potential Research Articles

Soluble HLA-G (sHLA-G) in human embryo culture media: a useful marker for assessing embryo quality, and implantation potential for IVF

Objective: Soluble HLA-G (sHLA-G) is known to be a critical modulator of local maternal-fetal immunological relationships during pregnancy. The detection of sHLA-G in embryo culture media suggests that sHLA-G may have a role in evaluating embryo quality and implantation potential after IVF. Therefore, we compared sHLA-G molecule expression in the media bathing individual embryos selected for transfer 72 hrs post fertilization by intracytoplasmic sperm injection (ICSI) with implantation and clinical pregnancy rates. We also compared sHLA-G molecular expression between morphologically “poor” and “good” quality embryos. Design: Retrospective cohort study Setting: Multi-site, private practice Patients: Infertile women 28–43 years old (n = 30) with own oocytes undergoing IVF. Interventions: Thirty women undergoing in vitro fertilization (IVF), all of whom had at least 2 embryos available for transfer, were included in the study. All oocytes were fertilized by ICSI and cultured individually in a 50 μl of P-1 (Irvine Scientific) media for 60–67 hr. The embryos were evaluated for embryo transfer (ET), whereupon they were transferred into 50 μl of Blastocyst (Irvine Scientific) media for ET with each embryo being further cultured for an additional 72 hr. Thereupon, 20–30 μl of P-1 medium was collected from the media in which the individual embryos were cultured. A specific sHLA-G ELISA monoclonal anti-HLA-G antibody and W6/32 anti-class I antibody was used in a sandwich ELISA assay to detect for the presence of sHLA-G in each individual sample. Culture media from trophoblast-derived JEG-3 cell line was utilized as a positive control in order to assess the specificity of the ELISA. Primary measures of outcome: The level of sHLA-G expression in each individual sample of P-1 medium relative was correlated with embryo quality as assessed on day 3 post fertilization using the Graduated Embryo Scoring (GES) method, and with subsequent pregnancy and implantation rate. Results: We tested for sHLA-G antigens expressed in the media surrounding each individual embryo. Embryos were classified in three groups based on such ranges. In Group I, the culture media of all embryos with a GES of 20–50 that were <7cells cleaved following 72 hrs in culture, poor sHLA-G expression (<0.120±0.017). No pregnancies occurred in this group. Group IIcomprised embryos that had attained 7–9 cells and had a GES of comprised embryos that had attained cells and had a of 70–100, but demonstrated weakly positive sHLA-G expression (mean 0.237±0.051) in the media. No pregnancies occurred in this group. Group III, embryos comprised those that reached to the 7 to 9 cell stage and each had a GES score of 70–100, but in addition showed strongly positive sHLA-G expression (mean 0.246±0.045). Six (20%) out of thirty patients had strongly positive sHLA-G expressing embryos on day 3. The clinical pregnancy and implantation rates following the transfer of these embryos were 84% (5/6) and 43% (9/21) per embryo, respectively. In addition, there was a positive correlation between the amount of sHLA-G in the culture media and the GES as well as the implantation rate per embryo. None of the atretic, arrested, or abnormally fertilized embryos revealed any sHLA-G in the media. Conclusions: The presence and concentration of sHLA-G in the culture medium 72 hours following fertilization by ICSI could provide a useful indicator measure of subsequent embryo implantation potential.

Optimal fertilization window in humans is about 4 hours

Objective: Although oocytes can be fertilized using ICSI as late as 24 hours after retrieval it has been well established that late fertilization does not lead to viable pregnancy. At the same time literature data suggest that ICSI performed at any time within 12 hours after oocytes harvesting yields the same pregnancy rates. Ironically, the common weakness of studies on the impact of ICSI timing on treatment outcome is a vaguely defined timing. Other weaknesses include long period of time for the study, allowing some additional factors to come into play, such as personnel changes and finally the number of cases. We report here to the best of our knowledge, the largest study to date that extends only over a period of 6 month and that has sharply defined timing. Design: The timing of ICSI was retrospectively correlated with ART treatment outcomes. Materials and Methods: Only ICSI cases were included in the study. TESE and TESA cases were excluded. Timing was recorded by an assisting person immediately at the start of the injection. In order to be assigned to the specific group the injection had to be started within the given time-interval and completed within 20 minutes. Results: Our data indicate that ICSI performed earlier than 2 hours or later than 4 hours after retrieval results in fewer embryos able to implant. The likely explanation for lower number of viable embryos after ICSI performed earlier than 2 hours after aspiration is relative immaturity of the oocytes, which could be indirectly confirmed by significantly lower fertilization rates. The reasons for lower implantation potential of embryos generated after ICSI performed more than 6 hours after aspiration need more investigation. Once implanted, embryos have the same chance to continue development regardless of the timing of injection. This would indicate that some sort of metabolic deficiency, rather than chromosomal imbalance is responsible for observed decline in implantation after 6 hours. Tabled 1 Conclusion: ICSI must be performed between 2 and 6 hours after oocytes retrieval to generate embryos with the highest developmental potential.

Improved outcome of frozen-thawed blastocyst transfer with Menezo’s 2-step thawing protocol

Improved outcome of frozen-thawed blastocyst transfer with Menezo’s 2-step thawing protocol

Using the male gamete for assisted reproduction: past, present, and future.

Using the male gamete for assisted reproduction: past, present, and future.

Day 3 versus day 5 embryo transfer: a prospective randomized study

Transfer of embryos at the blastocyst stage has been associated with exceptionally high implantation rates. There are, however, only a few prospective randomized studies comparing day 3 versus day 5 embryo transfer. Furthermore, the number of embryos replaced in the day 3 group transfer is often higher than the number of blastocysts replaced, thereby affecting implantation rates. A total of 118 patients undergoing standard IVF/intracytoplasmic sperm injection who had developed at least three 8-cell embryos showing <20% extracellular fragmentation on day 3 were randomized for day 3 or day 5 transfer. A maximum of two embryos were replaced. In this prospective, randomized study the implantation and pregnancy potential of embryos transferred on day 3 or day 5 were compared. Equal numbers of embryos were replaced in the two groups. There was no statistically significant difference between day 3 and day 5 transfer regarding positive human chorionic gonadotrophin rates (70 versus 67%), clinical pregnancy rates (61 versus 51%), implantation rates (44 versus 37%), twinning rates (42 versus 41%) and rates of early pregnancy loss (15 versus 29%). Transfer of embryos on day 3 or 5 showed similar implantation rates when equal numbers of embryos were transferred. Embryo transfer at the blastocyst stage seems to have no advantage over day 3 transfer in patients with more than two 8-cell embryos showing less than 20% fragmentation on day 3.

A prospective randomized study comparing day 3 with blastocyst-stage embryo transfer

A prospective randomized study comparing day 3 with blastocyst-stage embryo transfer

The effect of the biopsy procedure and the removal of a single blastomere from day 3 human embryos on their implantation potential.

Objective: Removal of a single blastomere for preimplantation genetic diagnosis (PGD) is a well established method; however, it is not known whether the biopsy procedure has no negative influence on the implantation potential of these manipulated embryos in humans. Possible alterations of implantation potential has no immense importance for many PGD indications, however, PGD for aneuploidy screening should sum up in improved implantation rate. The aim of the present study, therefore, was to investigate the effect of the removal of a single blastomere on the implantation potential of embryos in patients who were eligible for aneuploidy screening because of their age.

Embryo selection for transfer based only on pronuclear parameters alone but not in combination with day-3 embryo evaluation results in higher pregnancy and implantation rates.

Objective: Different studies have shown a relationship between various aspects of two-pronuclear (2-PN) morphology and the developmental capacity of embryos. However, it is not yet clarified whether pronuclear parameters can also predict the implantation potential of the developing embryos. The purpose of the present study, therefore, was to investigate the predictive value of pronuclear parameters on pregnancy and implantation rates. Design: A prospective and randomized study involving 290 ICSI-IVF patients. Embryos of 98 patients were selected for embryo transfer (ET) only on the bases of day-3 morphological evaluation. Embryos of 95 patients for ET were selected only on the bases of 2-PN evaluation while embryos of 97 patients were selected taking into account both the 2-PN and the day-3 morphological evaluation. Materials/Methods: Controlled ovarian hyperstimulation was carried out using the GnRHa (leuprolide acetate) long protocol and recombinant FSH in combination with hCG. Oocytes and embryos were cultured in groups using IVF-20 medium (Vitrolife). Evaluation of 2-PN stage was performed 15 to 17 hours after ICSI. Equal sized and centrally located pronuclei with polarized nucleoli (minimum 5 and maximum 18 in the two pronuclei together with a difference of maximum 4) in both pronuclei were the positive selection criteria at 2-PN stage evaluation. On day 3, embryos with more (and equal sized) blastomeres and with less fragmentation (and preferentially no multinucleation in the cells) were selected whenever possible for ET. In the last group (combination evaluation) 2-PN and day-3 morphological evaluation criteria were taken into account together. Embryo transfer was performed on day 3 using Wallace catheter. Clinical and laboratory data were analyzed and compared using One-way ANOVA or Kruskall-Wallis and the chi-square tests whenever appropriate. Results: There were no significant differences found in any of the clinical or laboratory parameters between the three groups. The most important patients’ parameters and results in the three groups are presented in the table. Implantation and pregnancy rates in the three groups. legendMethod of embryo selection for ETlegendP < 0.05 for clinical pregnancy and implantation rates between group-3 vs. group-1 and group-2.CyclesAge of women (±S.D.)Fertilization % (±S.D.)Embryos transferred (±S.D.)Clinical pregnancies (%)Implantation rateDay 3 morphology9836.3 (6.0)77.6 (20.5)3.6 (1.8)25 (25.5)15.12 PN morphology9536.1 (5.0)72.5 (23.0)3.3 (1.7)22 (23.2)12.12 PN + Day 3 evaluation9736.1 (5.8)72.5 (23.4)3.4 (1.7)38 (39.2)21.1legend P < 0.05 for clinical pregnancy and implantation rates between group-3 vs. group-1 and group-2. Open table in a new tab Conclusions: The results of the present study suggest that 2-PN (pronuclear stage) evaluation to select embryos for transfer is similarly efficient to day-3 morphological evaluation in predicting implantation potential of embryos. However, it is also demonstrated that 2-PN stage parameters are not associated closer than day-3 morphological criteria to predict implantation and pregnancy rates. On the other hand, applying 2-PN evaluation criteria additionally to day-3 morphology scoring to select embryos for transfer results in higher clinical pregnancy and implantation rates.

Does pronuclear morphology and/or early cleavage rate predict embryo implantation potential?

A total of 340 patients referred for in-vitro fertilization was included in a retrospective, comparative study in which zygotes were studied regarding alignment and polarization of nucleolar precursor bodies (NPB) and also early cleavage in relation to implantation and pregnancy rates for the 680 transferred embryos. At assessment of the pronucleus 18–19 h after sperm injection, NPB were checked for alignment/polarization. Twenty-six hours after sperm insemination the zygotes were assessed for early cleavage. At embryo transfer the two embryos with the best morphological score, irrespective of polarization and early cleavage, were selected for transfer. The overall rate of positive HCG tests 17 days after embryo transfer was 42% and the implantation rate 23%. Fourteen percent of the patients received two embryos with polarized NPB, with a positive HCG test of 51%. Embryo transfer with early-cleaved embryos was carried out in 21% of the cycles, with a pregnancy rate of 45%. Embryos with polarized NPB and/or early cleavage were transferred in 34% of the cycles, with a pregnancy rate of 51%, compared with a pregnancy rate of 38% when none of the embryos fulfilled these criteria (P-value 0.02). In this study the pregnancy rate was significantly higher when one or two embryos were polarized and/or early cleaved. It is concluded that in a cohort of morphologically good embryos, assessment for alignment/polarization of NPB and/or early cleavage can, together with conventional morphological criteria, serve as a simple non-invasive method for selection of embryos with high implantation potential.

Early Cleavage of Embryos to Two-Cell Stage Is Associated With Improved Progression to Blastocyst Stage, Implantation and Pregnancy Rates

Objectives: Recently embryos that cleave early were found to result in higher pregnancy rates. In the present study we compared the progression of the cleavage stage embryos to the blastocyst stage, implantation, pregnancy and abortion rates between cycles with and without early cleaved embryos.Design: Prospective study.Materials and Methods: Embryo cleavage to the two-cell stage approximately 27 hours after insemination was defined as early cleavage. Early cleavage was observed in 48 of 78 cycles. Excess embryos were left in sequential media to observe the rate of blastocyst formation.Results: Implantation and pregnancy rates were significantly higher in the early cleavage group. Excess embryos derived from the pool showing at least one embryo with early cleavage progressed to the blastocyst stage at a significantly higher rate. Tabled 1No early cleavageEarly cleavagePNo of cycles3048Mean age of patients34.233.1NSMean no of oocytes11.912.8NSFertilization rate74.877.3NSEarly two cells025.6<0.05Clevage rate95.996.8NSNo of embryos transferred3.94.0NSImplantation rate5.117.9<0.01Clin pregnancy rate16.7%47.9%<0.05Abortion rate20%13%NSBlastocyst progression25%56.7%<0.05 Open table in a new tab Conclusion: These findings suggest that early clevage of embryos can be used as good marker of implantation potential of cleavage stage embryos. Furthermore, sibling embryos in the pool of embryos showing early cleavage progress to the blastocyst stage at a higher rate suggesting that the potential of the embryo is evident from the early stages of development. Objectives: Recently embryos that cleave early were found to result in higher pregnancy rates. In the present study we compared the progression of the cleavage stage embryos to the blastocyst stage, implantation, pregnancy and abortion rates between cycles with and without early cleaved embryos. Design: Prospective study. Materials and Methods: Embryo cleavage to the two-cell stage approximately 27 hours after insemination was defined as early cleavage. Early cleavage was observed in 48 of 78 cycles. Excess embryos were left in sequential media to observe the rate of blastocyst formation. Results: Implantation and pregnancy rates were significantly higher in the early cleavage group. Excess embryos derived from the pool showing at least one embryo with early cleavage progressed to the blastocyst stage at a significantly higher rate. Tabled 1No early cleavageEarly cleavagePNo of cycles3048Mean age of patients34.233.1NSMean no of oocytes11.912.8NSFertilization rate74.877.3NSEarly two cells025.6<0.05Clevage rate95.996.8NSNo of embryos transferred3.94.0NSImplantation rate5.117.9<0.01Clin pregnancy rate16.7%47.9%<0.05Abortion rate20%13%NSBlastocyst progression25%56.7%<0.05 Open table in a new tab Conclusion: These findings suggest that early clevage of embryos can be used as good marker of implantation potential of cleavage stage embryos. Furthermore, sibling embryos in the pool of embryos showing early cleavage progress to the blastocyst stage at a higher rate suggesting that the potential of the embryo is evident from the early stages of development.

The Role of pFSH therapy for the male partner in cases of non-obstructive azoospermia

Objective: In our previous study we treated male partners suffering from severe OTA with prolonged pFSH therapy prior to IVF-ICSI cycles and presented better implantation potential of IVF-ICSI embryos after Metrodin therapy for 60–90 days. In this presented study we utilized our experience in azoospermic males suffering from non-obstructive azoospermia and presenting poor results in terms of testicular sperm retrieval and low fertilization potential and pretreated those patients with pFSH prior their next IVF-ICSI surgical sperm extraction cycle.

Autologous cumulus cell coculture improves embryo quality and blastocyst growth of icsi-derived human embryos

Intracytoplasmic sperm injection (ICSI) is commonly used to treat severe male factor infertility and results in a high fertilization rate. As with standard IVF embryos, ICSI-derived embryos vary in quality and implantation potential. Since embryo quality and implantation potential are two rate-limiting factors of IVF success, alternative culture systems, such as blastocyst culture and coculture systems, have been developed and implemented. We have recently reported that a simplified coculture system using the patients’ own cumulus tissue results in improved embryo quality and implantation rates for embryos fertilized using standard microdroplet insemination. In this study, we have evaluated autologous cumulus cell coculture in embryos resulting from ICSI. Embryo quality, selection for embryo transfer, and blastocyst development of nontransferred embryos was evaluated in 303 embryos fertilized by ICSI (303/402 [75.4%]). Following ICSI, oocytes were placed in individual 50 μL drops of HTF medium and randomly supplemented with cumulus tissue dissected from the oocytes prior to treatment of the oocyte/cumulus mass with trypain. Embryo quality was evaluated at 72 hours post–oocyte harvest. Percent blastocyst development of nontransferred embryos was determined at 144 hours post–oocyte harvest. The mean embryo quality score was significantly (P<.05) higher for embryos cultured with cumulus tissue (5.1 ± 0.21) than those cultured without cumulus (4.3 ± 0.23). Additionally, 64.1% (84/131) of the coculture embryos were selected for transfer compared to 43.6% (75/172) for standard ICSI embryos (P<.05). Lastly, nontransferred embryos continued to the blastocyst stage at a higher rate (P<.05) when cocultured (18/47 [38.3%] vs. 19/97 [19.6%]). These data indicate that coculture of ICSI embryos with autologous cumulus tissue results in improved embryo quality and developmental potential. Autologous cumulus culture is an advantageous coculture system due to its ease and safety. These data confirm the benefit of the system in ICSI-derived embryos.

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 comparative analysis of embryo implantation potential in patients with severe teratozoospermia undergoing in-vitro fertilization with a high insemination concentration or intracytoplasmic sperm injection

The objective of this study was to assess fertilization, implantation and pregnancy rates in infertile patients with severe teratozoospermia [P (poor prognosis) pattern sperm morphology assessed by strict criteria] treated by in-vitro fertilization (IVF) using a high insemination concentration (HIC), or by intracytoplasmic sperm injection (ICSI). This was a retrospective cohort study performed in an academic tertiary institution. The outcome of 115 consecutive ICSI cycles was compared to that of a similar number of cycles of IVF with HIC performed during a similar time frame and matched by woman's age and basal serum (cycle day 3) follicle stimulating hormone concentrations. The inclusion criteria were sperm morphology </=4% normal forms (P pattern) and >/=1x10(6) total motile spermatozoa per ejaculate. The diploid fertilization rate in the HIC-IVF group was 86% and in the ICSI group 68% (P < 0.05). Importantly, an equal number of embryos was transferred to both groups of patients. The morphological quality of the embryos (proportion of transfers having superior morphology embryo scores) was significantly better in the ICSI group than in the patients receiving HIC-IVF. Although there was a clear trend for better implantation and pregnancy rates in the ICSI group, these differences were not statistically significant. We conclude that, although HIC-IVF resulted in a higher fertilization rate than ICSI in patients with severe teratozoospermia, ICSI produced a significantly higher proportion of morphologically superior embryos with a tendency towards a higher implantation potential. Therefore, teratozoospermic patients having adequate numbers of motile spermatozoa should be offered ICSI as an alternative to modified (HIC) IVF treatment.

Fertilization and early embryology: Embryo quality and pregnancy potential of fresh compared with frozen embryos—is freezing detrimental to high quality embryos?*

To determine the effect of cryopreservation on embryo quality and the pregnancy potential of embryos, donated oocytes from the same donor (n = 24) were randomly allocated, with subsequent transfer to two or more different ovum recipients resulting in at least one fresh and one frozen embryo transfer cycle from the same cohort of oocytes. Endometrial receptivity was controlled in all ovum recipients, and male factor patients were excluded. The number of embryos transferred, mean embryo grade transferred, number of high quality embryos (grade < or = 2.5, grade 1 being best) transferred and embryo implantation and live birth rates are reported. Significantly more embryos (4.4 +/- 1.2 versus 3.3 +/- 1.2, P < 0.00003) of higher quality (1.9 +/- 0.5 versus 2.1 +/- 0.5, P < 0.013) and of a more advanced cell stage (3.0 +/- 0.6 versus 2.6 +/- 0.7, P < 0.019) were transferred fresh than after cryopreservation respectively. Implantation rates/embryo [19/151 (12.6%) and 9/111 (8.1%)] and live birth rates/transfer [11/42 (26.2%) and 6/45 (13.3%)], from fresh and frozen transfers respectively, were not significantly different despite the larger number of high quality embryos transferred fresh. Embryo cryopreservation adversely affects embryo quality, but does not have detrimental effects on the implantation or pregnancy potential of high quality embryos. Because of the loss of embryos during freeze-thawing during frozen embryo cycles, every effort should be made to attempt a fresh transfer.

Implantation potential and fetal viability of mouse embryos cultured in media supplemented with platelet-activating factor

Supplementation of culture media with PAF failed to increase the proportion of 2-cell mouse embryos which developed through to the expanded blastocyst stage after 72 h of culture in vitro. Culture of embryos with 1.86 microM-PAF, however, significantly increased the number of cells in expanded blastocysts. Culture in vitro with 0.186 or 1.86 microM-PAF significantly increased the ability of embryos to implant upon transfer to pseudopregnant recipients. The specificity of this effect was demonstrated by its inhibition by the specific PAF-receptor antagonist, SRI 63-441. Of the embryos which implanted, there was no difference in the proportions of PAF-treated or control embryos which resorbed nor was there any effect of PAF on fetal weight or crown-rump length measurements. Embryos cultured with 1.86 microM-PAF did have slightly heavier placentae than their respective controls. These results demonstrate the ability of exogenous PAF to enhance the viability of preimplantation mouse embryos cultured in vitro and support the hypothesis that a major site of the action of PAF during early pregnancy is the embryo itself.