A comparison of morphokinetic markers predicting blastocyst formation and implantation potential from two large clinical data sets.

PURPOSE: To demonstrate whether the standard morphokinetic markers used for embryo selection have a similar relationship to blastocyst formation and implantation in two large clinical data sets. METHODS: This is a retrospective cohort analysis striving to answer two distinct questions utilizing data sets from two large IVF clinics. Blastocysts (BL) and implanted blastocysts (I) in both clinics, IVI-Valencia (BL = 11,414, I = 479) and WMC (BL = 15,902; I = 337), were cultured in a time-lapse system (EmbryoScope, Vitrolife, Sweden). The study was designed to assess the relationship between early morphokinetic hallmarks and BL development, with a secondary analysis of implantation rates following single-embryo day 3 and day 5 transfers. RESULTS: We performed a detailed graphical analysis for t3, t5, duration of the second cell cycle (cc2) (t3-t2), and the ratio (t5-t3)/(t5-t2). The t5 timing was not affected between the clinics. However, Weill Cornell Medicine's (WCM) proportions were significantly affected by having BL vs. not. A significant decrease of blastocysts with longer t5 in WCM data, while t5 was more informative in the IVI data set for the implantation rate. CONCLUSIONS: Morphokinetic intervals for early cleavages were distributed differently between the clinics. Incorporation of embryo-selection algorithms depends on the individual clinic's selected developmental hallmarks, all of which must be validated before incorporation into clinical practice.

A rationale for biopsying embryos reaching the morula stage on Day 6 in women undergoing preimplantation genetic testing for aneuploidy.

STUDY QUESTION: Is there a benefit to assessing ploidy in delayed embryos reaching the morula stage on Day 6 of development? SUMMARY ANSWER: Day-6 morulae should be considered for biopsy in women <40 years old undergoing preimplantation genetic testing for aneuploidy (PGT-A) because they are associated with acceptable, albeit reduced, euploidy and implantation rates (IRs). WHAT IS KNOWN ALREADY: Embryo development and morphology have been shown to correlate with aneuploidy and pregnancy rates. During PGT-A cycles, embryos are biopsied if they reach the blastocyst stage by Day 5 or 6, whereas slow-developing embryos are typically deselected and discarded. Determining the viability of slow-developing embryos is particularly relevant for women undergoing PGT-A who have diminished ovarian reserve and a relatively low blastocyst yield. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study that was performed at an academic medical center. Patients who underwent IVF with PGT-A were reviewed for inclusion. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1615 cycles were reviewed. All cycles which involved a biopsy of a cavitating or compacted morula on Day 6 were included (n = 763). PGT-A was performed using array comparative genomic hybridization. The aneuploidy and implantation of morulae were compared to those of blastocysts originating from the same couples. MAIN RESULTS AND THE ROLE OF CHANCE: The study included 763 cycles in which 1260 morulae and 3014 blastocysts were biopsied. Women were divided into four age groups (<35, 35-37, 38-39 and ≥40 years): the prevalence of aneuploidy was consistently lower among blastocysts (40.3, 50.8, 56 and 78.3%, respectively) than among compacted morulae (68.7, 75.5, 88.9 and 98.1%, respectively) and cavitating morulae (57, 66.4, 81 and 91.6%, respectively) throughout the different age groups (P < 0.001). Of note, the majority of compacted morulae (98.1%) and cavitating morulae (91.6%) were aneuploid in women aged ≥40 years. Compacted and cavitating morulae had significantly higher rates of complex aneuploidy, which involves ≥3 chromosomes, compared with blastocysts (P < 0.001). Furthermore, euploid morulae were associated with a significantly lower IR (28.2 versus 54.6%; P = 0.002) and live birth rate (23.1 versus 55.0%; P = 0.001) compared to euploid blastocysts. LIMITATIONS REASONS FOR CAUTION: This study confirms that Day-6 morulae should not be discarded in young women undergoing PGT-A. However, a potential drawback of biopsying embryos at the morula stage is the inability to distinguish between inner cell mass and trophectoderm cell origin. The sample size of euploid morula transfer cycles in this study was limited. Thus, a larger cohort would be beneficial to validate the reassuring live birth and spontaneous abortion rates reported here. Furthermore, the reproducibility of our findings should be determined at different centers. WIDER IMPLICATIONS OF THE FINDINGS: Although Day-6 morulae are associated with higher aneuploidy rates and lower IRs compared to blastocysts, they still yielded successful pregnancies. Therefore, testing Day-6 morulae should be considered, especially for women <40 years old who are undergoing PGT-A with a small cohort of available blastocysts for biopsy. STUDY FUNDING/COMPETING INTEREST(S): The authors have nothing to disclose. They received no specific funding for this work. TRIAL REGISTRATION NUMBER: N/A.

Cryopreservation of murine embryos, human spermatozoa and embryonic stem cells using a liquid nitrogen-free, controlled rate freezer.

A Stirling Cycle Cryocooler has been developed as an alternative to conventional liquid nitrogen controlled rate freezers. Unlike liquid nitrogen systems, the Stirling Cycle freezer does not pose a contamination risk, can be used in sterile conditions and has no need for a constant supply of cryogen. Three types of samples from two species (murine embryos, human spermatozoa and embryonic stem cells), each requiring different cooling protocols, were cryopreserved in the Stirling Cycle freezer. For comparison, cells were also frozen in a conventional liquid nitrogen controlled rate freezer. Upon thawing, the rates of survival of viable cells were generally greater than 50% for mouse embryos and human embryonic stem cells, based on morphology (mouse embryos) and staining and colony formation (human embryonic stem cells). Survival rates of human spermatozoa frozen in the Stirling Cycle freezer, based on motility and dead cell staining, were similar to those of samples frozen in a conventional controlled rate freezer using liquid nitrogen.

Human zona pellucida micromanipulation and monozygotic twinning frequency after IVF.

To assess the association of zona pellucida micromanipulation and subsequent development of monozygotic twins, cases of assisted embryo hatching (AH) and intracytoplasmic sperm injection (ICSI) were identified and related to treatment type, implantation and zygosity data. Embryology records from all patients undergoing in-vitro fertilization (IVF) at this centre from January 1995 to March 1998 were reviewed. In this study, 3546 transfer cycles were completed, with clinical pregnancy established in 1911 (54% per transfer) patients undergoing a single IVF cycle. These pregnancies occurred in 1674 (88%) IVF cycles, 120 (6%) donor oocyte cycles (DER), and 117 (6%) frozen embryo transfer (FET) cycles. During the study period, 23 cases of monozygotic (MZ) twins were identified, representing an overall frequency of 1.2%. Chorionicity was determined by transvaginal ultrasound at 7 weeks when the number of embryos transferred was less than the number of fetal heart-beats, or when >1 fetal heartbeat per gestational sac was seen. Zygosity was confirmed by placental evaluation at delivery, and corroborated the antenatal diagnosis in all cases. Among IVF study patients the frequency of MZ twinning was not statistically different between zona manipulated and zona intact subgroups. While this investigation is the largest to date describing the relationship between MZ twins and zona procedures, studies with even greater statistical power are needed to clarify it more precisely, particularly in DER and FET settings. A greater overall frequency of MZ twinning for IVF patients may be a function of the higher number of embryos transferred in IVF, rather than discrete zona manipulations.

Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men.

The evident ability of the intracytoplasmic sperm injection (ICSI) procedure to achieve high fertilization and pregnancy rates regardless of semen characteristics has induced its application with spermatozoa surgically retrieved from azoospermic men. Here, ICSI outcome was analysed in 308 cases according to the cause of azoospermia; four additional cycles were with cases of necrozoospermia. All couples were genetically counselled and appropriately screened. Spermatozoa were retrieved by microsurgical epididymal aspiration or from testicular biopsies. Epididymal obstructions were considered congenital (n = 138) or acquired (n = 103), based on the aetiology. Testicular sperm cases were assessed according to the presence (n = 14) or absence (n = 53) of reproductive tract obstruction. The fertilization rate using fresh or cryopreserved epididymal spermatozoa was 72.4% of 911 eggs for acquired obstructions, and 73.1% of 1524 eggs for congenital cases; with clinical pregnancy rates of 48.5% (50/103) and 61.6% (85/138) respectively. Spermatozoa from testicular biopsies fertilized 57.0% of 533 eggs in non-obstructive cases compared to 80.5% of 118 eggs (P = 0.0001) in obstructive azoospermia. The clinical pregnancy rate was 49.1% (26/53) for non-obstructive cases and 57.1% (8/14) for testicular spermatozoa obtained in obstructive azoospermia, including three established with frozen-thawed testicular spermatozoa. In cases of obstructive azoospermia, fertilization and pregnancy rates with epididymal spermatozoa were higher than those achieved using spermatozoa obtained from the testes of men with non-obstructive azoospermia.

Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia.

OBJECTIVES: To provide fertility for men with nonobstructive azoospermia. METHODS: A retrospective review of treatment results at a university infertility center was undertaken. Sixteen couples entered an attempted in vitro fertilization (IVF)-intracytoplasmic sperm injection (ICSI) cycle for treatment of nonobstructive azoospermia. Each man was azoospermic, and the male factor diagnosis of nonobstructive azoospermia was made on testis biopsy for 14 men and on clinical grounds for 2 men. Sperm were retrieved by testicular biopsy on the day of oocyte retrieval. Results of testicular examinations, serum follicle-stimulating hormone levels, and testicular histology as well as evaluation of the success rates of sperm retrieval, fertilizations, and pregnancies were made. RESULTS: Sperm were extracted from testis biopsies in 10 of 16 (62%) testicular sperm extraction (TESE) attempts. For cycles in which sperm were retrieved, normal fertilizations were achieved for 51 of 98 (52%) mature oocytes injected with testicular sperm in 10 couples. Biochemical pregnancies were achieved for 6 of 16 (38%) couples, with clinical pregnancies during 5 of 16 (31%) attempts at sperm retrieval, and ongoing pregnancy and subsequent live delivery for 4 of 16 (25%) attempts. CONCLUSIONS; Pretreatment clinical parameters are unable to predict which men with nonobstructive azoospermia will have spermatozoa retrieved by TESE. When sperm are found, clinical pregnancies can occur for half (5/10) of these couples using TESE with ICSI, with ongoing pregnancy and delivery for 4 of 10 (40%). Many men with nonobstructive azoospermia will have retrievable sperm with testis biopsy that are suitable for ICSI; however, 6 of 16 (38%) couples will not have sperm retrieved with TESE and may undergo an unnecessary IVF procedure.

Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates.

This study was conducted to determine whether the mode of sperm immobilization prior to intracytoplasmic sperm injection (ICSI) influences fertilization by immature spermatozoa. Of the 837 ICSI cycles evaluated, 81 were performed with epididymal or testicular spermatozoa; 35 cycles with epididymal spermatozoa immobilized in the standard fashion resulted in fertilization and pregnancy rates of 48.3 and 51.4% respectively. When a more aggressive sperm immobilization technique (i.e. permanently crimping the sperm flagellum between the midpiece and the rest of the tail) was applied in 17 cycles, the resultant fertilization and pregnancy rates were significantly (P < 0.05) higher: 82.0 and 82.4% respectively. Similar increases in fertilization and ensuing pregnancy rates were also observed in ICSI cycles with the aggressive immobilization of frozen-thawed epididymal spermatozoa (eight cycles) versus standard immobilization (16 cycles). However, the fertilization rates for ICSI using testicular spermatozoa (five cycles) were basically the same, regardless of the immobilization technique. Furthermore, for ejaculated spermatozoa (756 cycles), the fertilization rates following aggressive sperm immobilization were also positively affected (73.4%), although no statistical differences in the clinical pregnancy rates were found. Because aggressive immobilization appears to affect sperm membrane permeabilization, the enhanced fertilization patterns observed in immature spermatozoa following aggressive immobilization may suggest a different membrane constitution in these spermatozoa. These findings indicate that immature gametes may require additional manipulation to enhance the post-ICSI events essential for adequate nuclear decondensation.