Comparison of pronuclear zygote morphology and early cleavage status of zygotes as additional criteria in the selection of day 3 embryos: a randomized study.

OBJECTIVE: Both pronuclear morphology and early zygote cleavage have been used in combination with day 3 criteria to predict implantation and pregnancy. However, in routine practice, it is impractical to use both these criteria to select embryos on day 3. The objective of the present study was to find which of the two criteria is more predictive in terms of implantation and pregnancy. DESIGN: Randomized study. SETTING: Hospital-based fertility center. PATIENT(S): A total of 330 IVF/ICSI patient cycles. INTERVENTION(S): Patients were randomized to two groups. The embryos of one group were classified into subgroups A, B, and C based on pronuclear morphology (group 1) and the embryos of the second group were classified into subgroups A, B, and C based on early cleavage status (group 2). MAIN OUTCOME MEASURE(S): Comparisons were made of implantation and pregnancy rates between groups 1 and 2, between subgroups within each group, and between the corresponding subgroups of groups 1 and 2. Progression of zygotes from day 1 to day 3 in group 1 was recorded, and retrospective analysis of pronuclear morphology of zygotes in group 2 was performed. RESULT(S): The overall implantation and pregnancy rates tended to be slightly higher for group 1 compared to group 2 patients, but not statistically significant. Further, there was no significant difference between the corresponding subgroups. Implantation and pregnancy rates of subgroup A zygotes from each group were significantly higher (P<.01) than the rates of subgroup C. CONCLUSION(S): There was no significant difference between the two groups in terms of implantation and pregnancy. However, pronuclear morphology is a more satisfactory criterion than early cleavage to assist embryo selection on day 3. This is because zygotes with early cleavage ability can be identified from their pronuclear morphology. Thus, observation for early cleavage on day 1 and assessment of progression of embryos on day 2 in addition to pronuclear morphology scoring is not necessary in the selection of embryos for transfer on day 3.

Beneficial effects of a higher starting dose of gonadotrophins in some poor responders.

We performed a prospective study to examine the benefits of higher starting dose in poor responders to gonadotrophins. One hundred eighty-seven normal responders (group 1), 40 poor responders (group 2), and 25 poor responders from group 2 (group 3) were included in the study. Groups 1 and 2 received 300 IU of metrodin HP for 5 days followed by 150 IU of recombinant human follicle-stimulating hormone (rhFSH) until the day before hCG. Group 3 received 450 IU of metrodin followed by rhFSH as in groups 1 and 2. Number of oocytes retrieved, rates of fertilization, implantation, pregnancy, and miscarriage rates were compared between the groups. There were no differences between the three groups in the fertilization rate. The higher dose of metrodin (450 IU) for 5 days increased the number of oocytes retrieved in some patients belonging to group 3 and significantly increased (P < 0.01) the implantation and pregnancy rates compared with group 2 patients. However, the higher dose of metrodin also increased miscarriage rates significantly in group 3 compared with groups 1 and 2 (P < 0.04). In some poor responders, a higher starting dose of gonadotrophin resulted in more oocytes retrieved and led to higher implantation and pregnancy rates and a higher miscarriage rate.

Cryopreservation of embryos by vitrification: current development.

Vitrification as a cryopreservation method has many primary advantages and benefits, such as no ice crystal formation through increased speed of temperature conduction, which provides a significant increase in cooling rates. This permits the use of less concentrated cryoprotectant agents so that the toxic effect is decreased. Additionally, chilling injuries are considerably reduced. Many variables in the vitrification process exist that can profoundly influence its effectiveness and the potential to improve the survival rates of vitrified cells. These include (i) the type and concentration of cryoprotectant (almost every kind of cryoprotectant is toxic), (ii) the temperature of the vitrification solution at exposure, (iii) the duration of exposure to the final cryoprotectant before plunging into LN2, (iv) the type of device that is used for vitrification (which influences the size of the vapor coat and cooling rate), and (v) the quality and developmental stage of embryos. Increasing the speed of thermal conduction and decreasing the concentration of cryoprotectant is an ideal strategy for cryostorage of embryos with vitrification methods. However, the actual rate of heat transfer during vitrification procedures may vary extremely depending on the device used, technical proficiency, and the specific movement at immersion. In addition, it is very important to mention that every cell has its own optimal cooling rate. To date, the "universal" vitrification protocol has yet to be defined. In light of this, it is important for researchers to achieve more consistent results from existing protocols and thereby to establish a standardized vitrification protocol that can be applied for cryopreservation of different developmental stages. Toward this end, it should be noted that vitrification protocols are starting to enter the mainstream of human ART. Protocols successfully applied for bovine oocytes and embryos have been used initially with human oocytes, and initial trials have been undertaken with human embryos and blastocysts, with births achieved. Vitrification is relatively simple, requires no expensive programmable freezing equipment, and relies on the placement of the embryos in a very small volume of vitrification medium that must be cooled at extreme rates not obtainable in regular enclosed cryostraws and cryovials. The more convenient protocols of ultrarapid freezing and vitrification, which eliminate the use of expensive controlled-rate freezers, await cross-over from use in other species, and they require validation from more extensive experimental study in humans. Despite this, the convenience of vitrification will push the development of this technique to higher levels of clinical efficiency and use.

A modified embryo cryopreservation method increases post-thaw survival with a concomitant increase in implantation.

OBJECTIVE: To achieve maximum post-thaw survival of frozen embryos. DESIGN: Historical controlled study. SETTING: Hospital-based fertility center. PATIENT(S): One hundred forty-five patients whose embryos were frozen and thawed according to the standard method, and 56 patients whose embryos were frozen and thawed according to a modified method. INTERVENTION(S): Modifications were made to the various steps of cryopreservation: freezing and thawing solutions, loading of embryos into the straws, and warming rates. MAIN OUTCOME MEASURE(S): Post-thaw survival, implantation, and pregnancy rates. RESULT(S): With the modified method, 138 (93%) of the 149 embryos thawed for 56 patients survived freezing, and 79.8% had all their blastomeres intact, which is almost double the result obtained (41.8%) for patients whose embryos were thawed with the standard method. The implantation and pregnancy rates were also significantly higher with the modified method compared with the standard method. CONCLUSION(S): Greater post-thaw embryo survival was achieved, with a concomitant increase in implantation and pregnancy rates, by modifying the various steps in the standard cryopreservation methodology. This has important implications in IVF practice.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Short coincubation of gametes in in vitro fertilization improves implantation and pregnancy rates: a prospective, randomized, controlled study.

OBJECTIVE: To examine the benefits of short coincubation of gametes compared to prolonged incubation in in vitro fertilization (IVF). DESIGN: Prospective randomized controlled study. SETTING: Hospital-based fertility center. PATIENT(S): One hundred thirty patients (group 1) and 129 patients (group 2). INTERVENTION(S): Oocytes from group 1 were exposed to spermatozoa for 2 hours, and oocytes from group 2 were exposed to spermatozoa for 20 hours. MAIN OUTCOME MEASURE(S): Fertilization and cleavage rates, embryo quality, and pregnancy and implantation rates were evaluated. Estradiol (E(2)) and progesterone (P(4)) levels were measured in the wells of culture dishes after 2-hour exposure of oocytes/zygotes to spermatozoa in group 1 and after 20-hour exposure in both the groups. RESULT(S): There was no difference between the two groups in the fertilization rate and the number of embryos obtained. However, the proportion of grade 1 embryos was significantly higher among group 1 compared to group 2 patients. Clinical pregnancy and implantation rates were significantly higher among group 1 compared to group 2. The significantly higher levels of E(2) and P(4) in the 20-hour cultures compared with the 2-hour cultures may have been detrimental to embryo quality, pregnancy, and implantation rates. CONCLUSION(S): Coincubation of gametes for 2 hours with standard or high insemination concentrations of spermatozoa significantly improved embryo quality and the pregnancy and implantation rates compared with overnight incubation of gametes.

Rescue ICSI of oocytes that failed to extrude the second polar body 6 h post-insemination in conventional IVF.

BACKGROUND: Attempts to 'rescue' by ICSI oocytes that remained unfertilized 24 h after conventional IVF have generally resulted in poor outcomes. The aim of the present study was to compare the outcome of rescue ICSI performed on one group of patients 6 h after initial insemination with those of another group where rescue ICSI was performed 22 h after initial insemination. METHODS: Twenty-five patient IVF cycles provided the oocytes for rescue ICSI 6 h after initial insemination, and 20 cycles provided the oocytes for rescue ICSI 22 h after initial insemination in this retrospective study. Fertilization and cleavage rates, embryo quality, implantation, and pregnancy rates after rescue ICSI were the main outcome measures. RESULTS: A fertilization rate of 70.3% was achieved with 6 h rescue ICSI compared with 48.5% with 22 h rescue ICSI (P < 0.0001). From 6 h rescue ICSI, 12 clinical pregnancies (48.0%) resulted in three sets of twins, eight singletons and one abortion. From 22 h rescue ICSI there was one (5.0%) singleton pregnancy and delivery of a healthy baby. Likewise, the implantation rate was 20.2% from 6 h rescue ICSI compared with 1.72% from 22 h rescue ICSI (P < 0.02). CONCLUSIONS: Rescue ICSI after 6 h post-insemination (46 h post-HCG) gave better fertilization, pregnancy and implantation rates compared with rescue ICSI after 22 h when oocytes have become aged.

Normal birth after microsurgical enucleation of tripronuclear human zygotes: case report.

Microsurgical enucleation of a single pronucleus from each of three tripronuclear zygotes was performed and the embryos were transferred to a 38-year-old woman on day 3 after fertilization. A normal healthy baby boy was born at 38 weeks and 4 days gestation, demonstrating that with polyspermic fertilization, removal of the extra male pronucleus allows the zygote to develop normally.