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.     

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.     

Limited value of morphological assessment at days 1 and 2 to predict blastocyst development potential: a prospective study based on 4042 embryos

BACKGROUND: Non-invasive and routine developmental markers are available to select the most viable embryo; however their respective values in terms of blastocyst development potential remain difficult to distinguish. METHODS: During this prospective study, the sequential growth of 4042 embryos individually cultured from day 1 to day 5/6 was recorded. Pronuclear morphology on day 1, and early cleavage, cell number and fragmentation rate on day 2 were evaluated for each zygote. Additionally, blastocyst transfers were analysed with regard to their implantation ability and early embryo development parameters. RESULTS: Once adjusted to each other, each of the four parameters remained related to blastocyst development. Early cleavage and cell number on day 2 were the most powerful parameters to predict the development of a good morphology blastocyst at day 5. Moreover, whereas transfers of a good morphology blastocyst were associated with high implantation and live birth rates, parameters of early development were not helpful in predicting their implantation ability. CONCLUSIONS: The combination of all four parameters allowed the prediction of blastocyst development with an area under the receiver operating characteristics curve of 0.688, which represents a fairly low prediction of embryo viability. Such results indicate that it is necessary to search for additional criteria, including the ability of the blastocyst to develop.     

Early cleavage is a valuable addition to existing embryo selection parameters: a study using single embryo transfers

BACKGROUND: To reduce the twin pregnancy rate, elective single embryo transfer (eSET) is increasingly implemented. Improvement of the results obtained with eSET can be achieved by better selection of the most viable embryo. This study investigated the predictive value of early cleavage (EC) as an additional parameter for selecting the embryo with the highest implantation potential by using data from SET's. METHODS: Data from 165 SET's were retrospectively evaluated. Cleavage to the 2-cell stage was determined 23-26 h after injection or 25-28 h after insemination. Selection of the embryo to be transferred was based on cell morphology and cell number on the day of transfer, not on the EC status. Additional information on the predictive value of EC on developmental potential was obtained by analysing 253 transfers with two embryos (double embryo transfer, DET) and blastocyst formation of 1160 surplus embryos. Logistic regression was used to determine the predictors of pregnancy or blastocyst development. RESULTS: A significantly higher pregnancy rate was observed after transfer of single EC embryos compared to single non-EC embryos (46 versus 18%). This result was confirmed by the significantly higher pregnancy rate after DET with two EC embryos as compared to DET with two non-EC embryos (45 versus 25%) and the blastocyst formation of EC embryos compared to non-EC embryos (66 versus 40%). Logistic regression showed that EC is an independent predictor for both pregnancy and blastocyst development in addition to cell morphology and cell number. CONCLUSIONS: In order to improve the selection of the embryo with the highest implantation potential, selection for transfer should not be based on cell number and morphology on the day of transfer alone, but also on early cleavage status.     

Early embryo cleavage is a strong indicator of embryo quality in human IVF.

BACKGROUND: In order to decrease multiple birth rates without decreasing birth rates overall, it is important to increase the capability of selecting the most optimal embryos for transfer. It has been shown that human embryos which cleave early, i.e. complete the first mitotic division within 25-27 h post insemination, provide higher pregnancy and implantation rates. METHODS AND RESULTS: In this prospective study, an evaluation of 10 798 scored embryos showed that early cleavage resulted in a significantly higher proportion of good quality embryos compared with late cleavage (62.5 versus 33.4%, P < 0.0001). When examining both day 2 and day 3 transfers together, early-cleaving embryos (306 transfers) gave rise to significantly higher rates of pregnancy/transfer (40.5 versus 31.3%, P = 0.0049), implantation (28.0 versus 19.5%, P = 0.0001) and birth/ongoing pregnancy (34.3 versus 24.0%, P = 0.0009) than did late-cleaving embryos (521 transfers). A stepwise logistic regression of all data showed that the total number of good quality embryos and female age were independent predictors of both pregnancies and birth. For intracytoplasmic sperm injection (ICSI) embryos, early cleavage was found to be an independent predictor of birth. CONCLUSIONS: Early embryo cleavage is a strong biological indicator of embryo potential, and may be used as an additional embryo selection factor for ICSI embryos.     

The effect of pronuclear morphology on embryo quality parameters and blastocyst transfer outcome.

BACKGROUND: Embryo quality may be accurately assessed as early as the pronuclear zygote phase, as shown in recent studies. However, it is not known whether good quality zygotes are destined to become good quality cleavage stage embryos and blastocysts. METHODS: In this retrospective study, 86 intracytoplasmic sperm injection-embryo transfer cycles were studied where each available embryo was scored from the zygote until the blastocyst stage. Embryonic normality parameters such as pronuclear pattern, early cleavage, cleavage stage embryo grade, the presence of embryos with > or =8 cells on day 3 and blastocyst quality were recorded. Embryo transfer was undertaken at the blastocyst stage and the outcome was studied according to the pronuclear pattern exhibited by the zygotes. RESULTS: Embryos that showed an ideal pronuclear pattern (0 PN pattern) cleaved earlier and faster and resulted in better quality cleavage stage embryos and blastocysts. The incidence of blastocyst formation was 72% in zygotes showing a 0 PN pattern, compared with 12.7% in zygotes with double pronuclear abnormality. Higher implantation and pregnancy rates were obtained when at least one blastocyst derived from a 0 PN pattern zygote was included in the set of embryos to be transferred. CONCLUSIONS: Our results indicate that the pronuclear pattern of the zygote is closely related to blastocyst formation and quality. Blastocysts derived from 0 PN zygotes have a higher potential for implantation.     

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.     

Day 3 embryo transfer with combined evaluation at the pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer

BACKGROUND: The respective advantages of day 3 and day 5 embryo transfer are a matter of debate. Previous comparisons did not include pronuclear stage zygote scoring and cumulative success rates (fresh and cryopreserved embryos). METHODS: Patients were randomized prospectively for day 3 or day 5 embryo transfer. Day 3 embryos were selected for transfer and cryopreservation by using combined evaluation at the pronuclear and cleavage stages. RESULTS: There was no difference between day 3 and day 5 fresh embryo transfers as to the rates of pregnancy (58 versus 62%), clinical pregnancy (56 versus 58%), delivery (50 versus 48%), implantation (35 versus 38%) and birth (33 versus 36%) rates. The corresponding values for cryopreserved embryo transfers were also similar. However, day 3 embryo transfer compared favourably with day 5 transfer when the pregnancy (90 versus 66%), clinical pregnancy (85 versus 62%) and delivery (77 versus 52%) rates were calculated per oocyte recovery attempt. CONCLUSIONS: With a selected population of good prognosis patients and our embryo selection criteria, the implantation potential of day 3 and day 5 embryos is equal. Per oocyte recovery attempt, day 3 transfer is more clinically efficient than day 5 transfer, but at least one transfer of cryopreserved embryos is necessary to manifest this superiority.     

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.     

Influence of group culture and culture volume on the formation of human blastocysts: a prospective randomized study.

The optimal culture conditions for embryos to reach the blastocyst stage are under investigation. One factor is the putative influence of autocrine or paracrine factors produced by the embryo itself. Studies in mice blastocysts showed a beneficial influence of micro-cultures and communal growth on pregnancy and implantation rates, attributed to these growth or survival factors. In humans, studies have only involved embryos up to day 2 or 3 without consistent results. Therefore a prospective, randomized study was designed to assess whether group culture or incubation volume would influence day 3 human embryos to develop into blastocysts. Embryos were initially cultured in groups until day 3, after which patients were randomly allocated to four groups. Group 1: group culture in a small volume; group 2: single culture in a small volume; group 3: single culture in a large volume; group 4: group culture in a large volume. No significant differences in blastocyst formation could be found between the four groups (35, 45, 36 and 36% respectively). Therefore any of the four modes can be used, whichever is most convenient. The only factor which made a statistically significant contribution was the number of embryos on day 3. An increase in the number of embryos by one embryo decreased the odds of obtaining a blastocyst by 6%. The overall pregnancy rate per transfer was 40%, and the implantation rate was 28%. Single culture in a small volume allows a direct and individual assessment of embryo morphology, and as such it seems preferable.     

Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development

BACKGROUND: It is of fundamental importance for IVF clinics to determine the most viable embryos for transfer. The challenge for ART clinics is to transfer fewer embryos, thereby minimizing the risk of multiple-infant births, while still maintaining the greatest chance of pregnancy for their patients. In this study, an investigation was made to determine if developmental markers on the day of fertilization (day 1) can predict good subsequent blastocyst development. METHODS AND RESULTS: A total of 1550 individually cultured 2PN embryos from 191 patients undergoing IVF/ICSI treatment at the Yale University Center for Reproductive Medicine and Infertility from February to December 2001 was included. The results showed a significant positive relationship between early-cleaving 2-cell embryos and subsequent good quality > or =4-cell, > or =7-cell and blastocyst development (P < 0.05). PN symmetry (the relative size of the PN to each other), when checked at the time fertilization, is also a significant indictor of good quality > or =4-cell, > or =7-cell stage embryos and blastocysts. Combined, a developing embryo showing PN symmetry with early cleavage and subsequent good > or =4-cell and > or =7-cell cleavage, has a one in two chance of developing into a good-quality blastocyst. CONCLUSION: Early embryo assessment can be used as an indicator of subsequent good blastocyst development.     

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.     

Effect of developmental stage of embryo at freezing on pregnancy outcome of frozen-thawed embryo transfer

BACKGROUND: The study aim was to investigate the impact of the developmental stage of embryos on pregnancy outcome of frozen embryo transfer (FET). METHODS: The survival rates of embryos after thawing and pregnancy outcome following FET were compared retrospectively between three cryopreservation strategies utilizing either zygote, day 2 or day 3 embryo freezing. RESULTS: A total of 4006 embryos was analysed in 1657 thaw cycles. The highest (P < 0.0001) survival rate (all cells survived) was observed for zygotes (86.5%), followed by day 2 (61.7%) and day 3 (43.1%) embryos. FET was performed in 1586 (95.7%) of all thaw cycles, resulting in overall clinical pregnancy and implantation rates of 20.7 and 14.2% respectively. The delivery rate per transfer was 16.5%, and live birth rate per transferred embryo 11%. There were no significant differences in clinical pregnancy, implantation, delivery and birth rates between frozen zygote, day 2 and 3 embryo transfers. However, an elevated miscarriage rate was observed in the day 3 group (45%) compared with zygotes (21.3%; P = 0.049) and day 2 embryos (18.3%; P = 0.004). The overall efficacy of FET (birth rate per thawed embryo) was 7.3%. The efficacy was lower in day 3 group (4.2%) than in the zygote (7.1%; P = 0.082) and day 2 (7.6%; P = 0.027) groups. CONCLUSIONS: The developmental stage of embryos at freezing has a profound effect on their post-thaw survival, but seems to have little effect on rates of clinical pregnancy, implantation, delivery and birth after FET. The elevated miscarriage rate for day 3 frozen embryo transfers may be caused by damage during freeze-thaw procedures. The low survival rate and elevated miscarriage rate were both responsible for a reduced overall efficacy for day 3 FET when compared with zygotes and day 2 embryos.     

The Graduated Embryo Score (GES) predicts blastocyst formation and pregnancy rate from cleavage-stage embryos

BACKGROUND: Embryo morphology and cleavage rates alone do not consistently identify embryos with high implantation potential following IVF. Blastocyst transfer has been reported to improve success rates by identifying potentially superior quality embryos. Algorithms for predicting IVF outcomes based on the presence of early developmental milestones have been proposed. Here we introduce the Graduated Embryo Score (GES). METHODS: Nucleolar alignment along the pronuclear axis, regular cleavage and degree of fragmentation at the first cell division, and cell number and morphology on day 3 were weighted to create a possible GES of 100 for each of 1245 fertilized embryos derived from 109 patients aged <40 years. The GES was correlated with IVF outcome. RESULTS: Of 983 embryos for extended culture, 349 (36%) developed to blastocyst and 180 (18%) were good quality (grade I-II). When ranked by cell number and morphology alone, 34% of embryos with > or =7 cells and <20% fragmentation formed good quality blastocysts. Using GES, embryos scoring 90-100 had 64% blastocyst formation compared with 31% scoring 70-85 and with 11% scoring 30-65. Embryos scoring 70-100 had 44% blastocyst development compared with 9% scoring 0-65. Fifty-six patients (51%) conceived on-going gestations from 294 transferred embryos. In patients with at least one transferred embryo scoring > or =70, the pregnancy rate was 59% compared with 34% if all embryos scored <70. The overall implantation rate was 28%. Among embryos scoring 70-100, an implantation rate of 39% was seen, compared with 24% among embryos scoring 0-65. CONCLUSIONS: Predicting which cleaved embryos will form blastocysts could permit the high success rates associated with blastocyst transfer to be achieved from day 3 embryo transfer.     

Avoiding multiple pregnancies in ART: a plea for single embryo transfer

It has been generally accepted that triplets after IVF/intracytoplasmic sperm injection (ICSI) can and should be avoided by adopting a standard strategy of replacing no more than two embryos. However, there is an increasing awareness of the risks and costs and of the epidemic size of twin pregnancies after IVF/ICSI. This has resulted in efforts to replace no more than one embryo. However, this approach has been hampered by our relative inability to identify embryos with a very high implantation potential. To identify such embryos, a number of strategies are being considered, both at the two pronuclear (2PN), early cleavage and the blastocyst stages. At the 2PN stage, the polarity characteristics of the nucleoli have been shown to be correlated with a high implantation rate. Similarly, the morphological characteristics at day 2 and 3 have been used to describe top quality embryos in approximately 75% of all IVF/ICSI cycles. Blastocyst culture has resulted in very high implantation rates in the hands of some authors. No approach has shown its superiority at present, but initial experience with single embryo transfer (SET) at the early cleavage stage by Scandinavian and Belgian groups shows that an ongoing pregnancy rate of 35% and more can be achieved. Proper identification of patients at risk of a twin pregnancy after double embryo transfer is equally important. It is clear that mainly young patients (aged <34 years) during their first, perhaps first two, IVF/ICSI cycles constitute the main population at risk (responsible for >80% of all twins) and are the main target group for twin prevention by SET of a top quality embryo at whatever stage. Therefore, in our opinion, although a further fine-tuning of both embryo and patient characteristics relating to a high risk for (twin) pregnancy is desirable, SET should be introduced carefully and progressively in each IVF/ICSI programme from now on. Correct counselling is very important and both public and private insurers will have to join in the discussion.     

Day 5 versus day 3 embryo transfer: a controlled randomized trial

Blastocyst transfer has been suggested to improve implantation rate without affecting pregnancy rate. The aim of this study was to compare the pregnancy and implantation rates of day 3 and 5 transfers in a prospective randomized manner. Patients with four or more zygotes were randomly allocated on day 1 to either day 3 or 5 transfers. Fertilization was achieved through regular IVF or intracytoplasmic sperm injection. Zygotes were kept in Medicult IVF medium for day 3 transfers and transferred into G1.2 and G2.2 on day 1 and 3 respectively for day 5 transfers. The morphologically best two or three embryos or blastocysts were chosen for transfer in both groups. Overall pregnancy rates per embryo transfer were the same (39%) in day 3 and 5 transfers. Implantation rates were 21 and 24% for day 3 and 5 transfers respectively. The pregnancy and implantation rates for day 5 transfers were significantly affected by the availability of at least one blastocyst to transfer and the number of zygotes. The number of good quality embryos on day 3 also significantly affected pregnancy and implantation rates on day 5 transfers. Multiple gestation rate, number of abortions and ongoing pregnancies were similar in both groups. In conclusion, day 3 and 5 transfer had similar pregnancy, implantation and twinning rates. Currently, day 5 transfers have no advantages over day 3 transfers.     

The predictive value of pronuclear morphology of zygotes in the assessment of human embryo quality

BACKGROUND: Recent studies have shown that zygote morphology could be used for the assessment of human embryo quality. Pronuclear (PN) morphology is based on certain distinct features seen in zygotes 16-18 h after fertilization. In the present study PN stage morphology was assessed and combined with a single embryo transfer in order to investigate whether currently used zygote classifications are able to predict embryo quality and implantation rates. METHODS AND RESULTS: Zygotes were analysed according to two different classification systems. In the first, a total of 764 zygotes was analysed according to the degree of polarization of nucleolar precursor bodies (NPB). Zygotes with unpolarized PN (i.e. scattered localization of NPB) showed significantly slower (P < 0.005) cleavage rates (38.9%) than zygotes having at least one pronucleus polarized (57.3% and 54%). However, there was no difference in the pregnancy rate in 105 single embryo transfers between the groups. The appearance of a cytoplasmic halo was related to embryo morphology. Embryos derived from halo-positive zygotes had significantly better (P < 0.05) morphology (60.9%) compared to halo-negative derived embryos (52.2%), but in terms of pregnancy rates no difference was found. A total of 1520 zygotes was analysed according to a second classification system, which was based on the number and distribution of NPB. In the comparative analysis, none of the six different classes produced superior quality embryos or higher pregnancy rates in 144 single embryo transfers. CONCLUSIONS: Our results indicate that there are no significant differences in embryo quality or implantation/pregnancy rates between proposed zygote classes.     

Zygote transfer on day 1 versus cleavage stage embryo transfer on day 3: a prospective randomized trial

BACKGROUND: It has been reported that pronuclear morphology is related to embryo quality and viability, and that zygote stage embryos might establish pregnancies after being transferred to the uterus. The objective of this study was to investigate whether transferring zygotes on day 1 would result in similar pregnancy rates compared to transferring cleavage stage embryos on day 3 in a prospective randomized trial. METHODS: Patients undergoing IVF/ICSI treatments were randomized to either day 1 or day 3 transfers by envelope withdrawal technique. Zygotes were classified as 'pattern 0' and 'non-pattern 0' according to the size and alignment of pronuclei, the number and distribution of nucleoli. The two best zygotes or embryos were transferred on day 1 or day 3 respectively. The primary outcome measure was pregnancy rate. RESULTS: Pregnancy rates were higher in day 3 group (55/131, 42%) when compared to day 1 (34/123, 28%, P = 0.024). Similarly, implantation rates were higher in day 3 group (P = 0.03). There were more cycles with cryopreservation in the day 1 group (P < 0.001). Embryo quality on day 3 was similar between pattern 0 and non-pattern 0 zygotes. CONCLUSIONS: Day 3 embryo transfers result in better pregnancy and implantation rates compared to day 1 zygote transfers. The present pronuclei scoring cannot reliably select zygotes for transfer on day 1.     

Pregnancies achieved after frozen-thawed pronuclear oocytes obtained by intracytoplasmic sperm injection with spermatozoa extracted from frozen-thawed testicular tissues from non-obstructive azoospermic men.

The use of frozen-thawed testicular tissue as a source of spermatozoa for intracytoplasmic sperm injection (ICSI) in non-obstructive azoospermia yields favourable fertilization and pregnancy rates while avoiding both repetitive biopsies and unexpected cycle cancellations. Spermatozoa were obtained from frozen-thawed testicular biopsy specimens from 67 non-obstructive azoospermic men. Following fertilization, supernumerary two pronuclear (2PN) oocytes were frozen. After thawing, 17 cycles of embryo transfer were carried out with a mean number of 2.7 embryos and a mean cumulative embryo score (CES) of 18.3 per transfer. The clinical pregnancy and implantation rates per transfer in these cycles (23.5 and 8.3% respectively) were comparable to those of fresh embryo transfers (35.7 and 12.7% respectively) with a mean number of 2.7 embryos and a mean CES of 28.7 per transfer. Abortion rates, although higher with cryopreserved 2PN oocytes were not significantly different. With this approach, cryopreservation of supernumerary 2PN oocytes can be used to improve the cumulative pregnancy rates in a severely defective spermatogenetic population. To our knowledge, these are the first pregnancies reported which have been obtained by the transfer of cryopreserved pronuclear oocytes obtained from ICSI using cryopreserved testicular spermatozoa.     

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.