Obstetric and neonatal outcomes after the transfer of vitrified-warmed blastocysts developing from nonpronuclear and monopronuclear zygotes: a retrospective cohort study.

OBJECTIVE: To evaluate the obstetric and neonatal outcomes after the transfer of vitrified-warmed single blastocysts developing from nonpronuclear (0PN) and monopronuclear (1PN) zygotes. DESIGN: Cohort study. SETTING: Affiliated hospital. PATIENT(S): This study was a retrospective analysis of 435 0PN and 281 1PN vitrified-warmed single blastocyst transfers, and 151 0PN and 75 1PN singletons, compared with 13,167 two-pronuclear (2PN) vitrified-warmed single blastocyst transfers and 4,559 2PN singletons, respectively. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pregnancy rate (PR), abortion rate (AR), live birth rate (LBR), and singleton birthweight were the primary outcome measures. RESULT(S): PR, AR, and LBR were similar when compared between the 0PN and 2PN groups after vitrified-warmed blastocyst transfer. However, the 0PN group had a higher birthweights, higher z scores, and a greater proportion of very large for gestational age newborns. When comparing the 1PN and 2PN groups, we found that the PR was similar whereas the AR was higher and the LBR was lower. No differences were detected in the other neonatal outcomes. CONCLUSION(S): The results of the present study show that the transfer of 2PN blastocysts should be prioritized because of a higher AR and a lower LBR after 1PN blastocyst transfers and a higher birthweight after 0PN blastocyst transfers when compared with 2PN blastocyst transfers. Our data indicate the need for concern about the safety of 1PN and 0PN embryo transfers.

Noninvasive embryo selection: kinetic analysis of female and male pronuclear development to predict embryo quality and potential to produce live birth.

OBJECTIVE: To evaluate a noninvasive method of examining euploid embryos, focusing on kinetic analyses, from second polar body extrusion to pronuclear membrane breakdown (PNMBD). DESIGN: Retrospective embryo cohort study. SETTING: Private IVF clinic. PATIENT(S): 213 frozen-thawed single blastocyst transfers. INTERVENTION(S): Fertilized oocytes were recorded by means of time-lapse photography, followed by kinetic analysis of female and male pronuclei (PNs). MAIN OUTCOME MEASURE(S): The differences in size between the 2PNs in embryos resulting in live births compared with those of embryos from failed pregnancies were analyzed according to sequential size from early PN stages to PNMBD. RESULT(S): It was found that the difference in areas between male and female PNs immediately before PNMBD is a better predictor of embryo quality if this difference is below a known cutoff value. The size of male PNs 8 hours before the onset of PNMBD should be larger than female PNs (B). The difference in size between male and female PNs 8 hours before PNMBD should be larger than the difference in their size immediately before PNMBD. When normal embryos were defined using the equation (A∪C)∩B, the birth rates for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were 68.1% and 50.0%, respectively. For the remaining embryos, defined as abnormal according to the above criteria, birth rates were 9.4% for IVF and 4.2% for ICSI. CONCLUSION(S): We have developed a method for noninvasive embryo evaluation by means of the kinetic analysis of female and male PN growths. This method should enable us to select embryos that have a higher potential for healthy births.

Identification of biparental and diploid blastocysts from monopronuclear zygotes with the use of a single-nucleotide polymorphism array.

OBJECTIVE: To select normal fertilized diploid blastocysts in patients who had only monopronucleated (1PN) embryos for transfer. DESIGN: Experimental study. SETTING: University-affiliated center. PATIENT(S): Couples who were undergoing intracytoplasmic sperm injection treatment and had 1PN blastocysts. INTERVENTION(S): In a preliminary test, limited cells of parthenogenetic human embryonic stem cells (phESCs) and normal fertilized blastocysts were analyzed with the use of a low-density single-nucleotide polymorphism (SNP) array to identify the distribution pattern and rate of heterozygosity. In the clinical application, 1PN blastocysts were analyzed with the use of the SNP array. Only diagnosed normal blastocysts were transferred. The diagnosed uniparental blastocysts were validated by imprinted gene expression. MAIN OUTCOME MEASURE(S): Distribution pattern and rate of heterozygosity between parthenogenesis and normal fertilization. RESULT(S): In the pretest, phESCs exhibited distinct distribution pattern and lower rate of heterozygosity, compared with normal fertilized blastocysts after SNP analysis. In particular, homozygous hESCs showed a panhomozygosity distribution pattern, hybrid phESCs showed a partial homozygosity distribution pattern, and normal fertilized blastocysts exhibited a panheterozygosity distribution pattern with an average of 20.21% heterozygosity rate; 13.6% was found to be the minimum cutoff to predict normal fertilized samples. In the clinical application, 24 1PN blastocysts were analyzed; 10/24 showed chromosomal abnormalities, 3/24 showed panhomozygosity with 0.45%-0.8% heterozygosity, and 1/24 showed partial homozygosity with 6.54% heterozygosity. The remaining 10 blastocysts, with a panheterozygosity distribution pattern and higher genomic heterozygosity rate, were diagnosed as normal-fertilization diploid embryos; three were transferred and resulted in two healthy newborns. CONCLUSION(S): The low-density SNP array might serve as a cost-effective method to identify biparental origin and diploid 1PN blastocysts for transfer.

¿Los parámetros morfocinéticos permiten predecir las aneuploidías embrionarias? / Do morphokinetic parameters allow the prediction of embryo aneuploidy?

ANTECEDENTES: La selección embrionaria sigue siendo uno de los grandes retos de la embriología para poder realizar más transferencias de un único embrión y mejorar los resultados de los tratamientos. Las aneuploidías son una de las principales causas que limitan las probabilidades de éxito, pero las técnicas disponibles actualmente para su diagnóstico son invasivas y costosas. El desarrollo de nuevos métodos precisos y no invasivos basados en parámetros morfocinéticos para detectarlas puede ser muy útil. OBJETIVOS: Revisar estudios relevantes que analicen la relación entre los parámetros morfocinéticos y las aneuploidías y el potencial de éstos para predecirlas. MATERIAL Y MÉTODO: Búsqueda en Pubmed utilizando palabras relacionadas con la morfología, la morfocinética y las aneuploidías embrionarias. También se han revisado las referencias de los artículos más relevantes y trabajos presentados en congresos internacionales celebrados recientemente. RESULTADOS: Numerosos estudios han analizado la capacidad de los parámetros morfocinéticos para detectar aneuploidías embrionarias. Algunos grupos han encontrado correlaciones significativas entre la ploidía y el tiempo de desaparición de los pronúcleos, los tiempos de diferentes divisiones celulares tempranas o parámetros más tardíos, como la duración de la compactación o el inicio de la blastulación. A partir de estos hallazgos, se han desarrollado modelos para clasificar los embriones según el riesgo de aneuploidía. Sin embargo, otros estudios no han encontrado ninguna correlación estadísticamente significativa y los resultados todavía son controvertidos. CONCLUSIONES: Ninguno de los parámetros morfocinéticos analizados proporciona suficiente precisión para diagnosticar aneuploidías embrionarias en las pacientes en las que está indicado realizar diagnóstico genético preimplantacional. Las diferencias entre los grupos pueden deberse al tipo de pacientes incluido y/o a las distintas técnicas utilizadas en cada laboratorio. Es necesario realizar más estudios que aclaren esta relación. Los parámetros morfocinéticos pueden, en determinados casos, ayudar a seleccionar embriones con mayor probabilidad de ser euploides, pero hoy en día la única forma fiable de hacer este diagnóstico es a través del diagnóstico genético preimplantacional. Ámbito: embriología. DISEÑO: revisión bibliográfica

BACKGROUND: Embryo selection still remains one of the great challenges in embryology in order to perform more single embryo transfers and improve clinical outcomes. Aneuploidy is one of the major causes of IVF failure, but the techniques applied for assessing embryo ploidy today are still invasive and expensive. New accurate non-invasive methods to select chromosomally normal embryos based on morphokinetic parameters can become useful. OBJECTIVES: To review relevant studies analysing the relationship between embryo morphokinetics and aneuploidy and the potential of these parameters to predict ploidy. MATERIAL AND METHOD: Search of Pubmed using keywords related to morphology, morphokinetics and embryo aneuploidy. References of the most relevant articles and communications presented in recent international meetings have also been reviewed. RESULTS: Numerous studies have analysed the ability of morphokinetic parameters to detect embryo aneuploidy. Some groups have found significant correlations between ploidy and timing of pronuclei fading, timing of early mitotic divisions as well as later morphologic events, such as the duration of compaction and the time of initiation of blastulation. Based on these results, different models have been developed to categorize the risk of aneuploidy in embryos. However, other studies have not found any statistically significant correlation and the results are still controversial. CONCLUSIONS: None of the morphokinetic parameters analysed is accurate enough to detect embryo aneuploidy in patients indicated for preimplantation genetic screening. Differences between groups may be due to the patient's populations included and/or variations in the techniques applied. Further studies are needed to clarify the possible relation. Morphokinetic parameters can aid in certain cases, to select embryos with higher probability of being euploid. However, preimplantation genetic screening still remains the only reliable technique to do such analysis. SETTING: embryology. DESIGN: review

Advanced scheduling for zygote intrafallopian transfer is possible via the use of a hormone replacement cycle for patients who have experienced repeated implantation failures.

PURPOSE: Zygote intrafallopian transfer (ZIFT) is an effective option for patients who have experienced repeated implantation failures (RIF) in assisted reproductive technology (ART) treatment. However, advance planning for the day of the operation can be problematic. Using a hormone replacement cycle (HRC) makes it possible to plan for the day of ZIFT. In the present study, we evaluated whether HRC-ZIFT is useful for RIF patients who have experienced difficulties obtaining morphologically good embryos in vitro. METHODS: A total of 55 patients with a history of five or more unsuccessful transfers received HRC-ZIFT between June 2008 and June 2013. The oocyte pick-ups were performed and the oocytes showing two pronuclei (2PN) were cryopreserved. After receiving more than five 2PN oocytes, the operation day was scheduled in advance, and as a consequence, a HRC was started and ZIFT was performed. The clinical outcomes were evaluated. RESULTS: The average age of the patients was 39.3 years, and the previous OPU and ET attempts numbered 7.5 and 6.9, respectively. The number of previously transferred embryos was 11.8, and the number of morphologically good embryos (MGEs) was only 1.2. The number of transferred 2PN oocytes was 6.7, and the subsequent pregnancy rate was 23.6 %. No ectopic or multiple pregnancies were observed, but there were 6 cases of miscarriage. CONCLUSION: Among RIF patients, in particular those who have difficulty obtaining MGEs in vitro, ZIFT might be a useful option. The HRC allows patients and medical staff to plan for the operation day in advance.

Zygote intrafallopian transfer among patients with repeated implantation failure.

OBJECTIVE: To summarize the experience of a single center with laparoscopic zygote intrafallopian transfer (ZIFT) performed exclusively among patients with high-order repeated implantation failure (RIF) following in vitro fertilization-embryo transfer (IVF-ET). METHODS: A retrospective cohort study was performed at the Edith Wolfson Medical Center, a tertiary referral university hospital located in Holon, Israel. A group of 176 patients with 8.15±3.9 previously failed IVF-ET cycles underwent 280 ZIFT procedures between 1995 and 2010. The main outcome measure was the live birth rate per patient treated. RESULTS: In all, there were 274 fresh and 6 frozen ZIFT cycles recorded in the study cohort, resulting in 96 clinical pregnancies per attempt (34.3%) and 72 live births (25.7%). The live birth rate per patient was 39.8%. CONCLUSION: The use of ZIFT remains a powerful tool in the clinical management of selected patients with high-order RIF. This procedure should be kept in mind when all other measures fail among patients with at least 1 unobstructed fallopian tube.

Successful pregnancy in vitrified/warmed blastocyst intrafallopian transfer.

OBJECTIVE: To analyze whether the use of blastocyst intrafallopian transfer is a feasible option in a case of repeated difficult ET. DESIGN: Case report. SETTING: Public hospital. PATIENT(S): Forty-year old nulliparous patient. INTERVENTION(S): Transfer of two vitrified/warmed blastocysts into the right tube by means of laparoscopy. MAIN OUTCOME MEASURE(S): Successful ET, clinical pregnancy. RESULT(S): Successful ET procedure resulting in positive ß-hCG and clinical pregnancy. CONCLUSION(S): In cases of repeated difficult ETs (regardless of whether the patient shows cervical adhesions or any type of genital malformations), blastocyst intrafallopian transfer can be a successful alternative approach.

Office microlaparoscopic intrafallopian transfer of day one zygote versus day three embryo transfer after previous failed ICSI trials.

The objective of the 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, using the office microlaparoscopic procedure. Patients undergoing IVF/ICSI treatments were randomized to either day 1 or day 3 transfers after previous failed ICSI trials due to failed implantation. The primary outcome measure was pregnancy rate. 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. Day 3 embryo transfers result in better pregnancy and implantation rates compared to day 1 zygote transfers.

Analysis of pronuclear zygote configurations in 459 clinical pregnancies obtained with assisted reproductive technique procedures.

BACKGROUND: Embryos selection is crucial to maintain high performance in terms of pregnancy rate, reducing the risk of multiple pregnancy during IVF. Pronuclear and nucleolar characteristics have been proposed as an indicator of embryo development and chromosomal complement in humans, providing information about embryo viability. METHODS: To correlate the zygote-score with the maternal age and the outcome of pregnancy, we analyzed the pronuclear and nucleolar morphology, the polar body alignment and the zygote configuration in 459 clinical pregnancies obtained by IVF and ICSI in our public clinic in Reggio Emilia, Italy. We derived odds ratios (OR) and Corenfield's 95% confidence intervals (CI). Continuous variables were compared with Student's t-test; P lower than .05 was considered statistically significant. RESULTS: We observed a significant increase of "A" pronuclear morphology configuration in 38-41 years old patients in comparison to that lower than or equal to 32 years old and a significant decrease of "B" configuration in 38-41 years old patients in comparison to that lower than or equal to 32 and in comparison to that of 33-37 years old. Related to maternal age we found no significant differences in P1 and in P2 configuration. We found no correlation between zygote-score, embryo cleavage and embryo quality. CONCLUSIONS: Our results confirm the limited clinical significance of zygote-score suggesting that it can not be associated with maternal age, embryo cleavage and embryo quality. The evaluation of embryo quality based on morphological parameters is probably more predictive than zygote-score.

Comparison of pregnancy and implantation rates in zygote intrafallopian transfer and uterine embryo transfer for nontubal infertility.

We carried out a prospective randomized trial on 220 couples with nontubal factor infertility to compare pregnancy rates and implantation rates after zygote intrafallopian transfer (ZIFT) and uterine embryo transfer (UET). The zygote was transferred by laparoscopy into the fallopian tube 24 hours after oocyst retrieval. UET was performed 72 hours after retrieval with abdominal sonography guide. Transfer was performed in 102 cycles in the ZIFT and 100 cycles in the U ET group. The pregnancy and implantation rates were significantly higher in the ZIFT group (42.1% and 11.7%) than in the UET group (21.0% and 7.8%) (P < 0.05). ZiFT could be considered for couples who have limited time and adequate financial support.

Mapping lineage using BAC-Cre reporter lines.

As the brain develops, progenitor cells acquire the features of specific neuronal or glial subtypes through dynamic expression of the fate-determining signaling molecules and their targeting transcription factors. An effective and versatile approach for tracing lineage of progenitors into adult cell types is to target the promoter of an interested gene with Cre (a phage DNA recombinase) to achieve simultaneous activation during neurogenesis. The bacterial artificial chromosome (BAC) is an efficient Cre carrier. Not only the targeted gene remains diploidy in BAC-Cre transgenic mice, but also the large portions of the gene's regulatory elements to be incorporated in the BAC allow Cre to sufficiently and reliably reproduce the endogenous gene expression pattern. When the BAC-Cre mouse is crossed to a Cre reporter mouse, even Cre is transiently expressed. Cre-loxP mediated recombination can permanently activate a reporter gene, such as green fluorescent protein (GFP) in all lineage cells of the gene. Experimental designs and procedures for RecA-based BAC DNA modification and preparation for pronuclear injection are highlighted. Suggestions for the use of BAC-Cre transgenic mice in fate-mapping analyses are also provided.

Comparison of different cryopreservation techniques: higher survival and implantation rate of frozen-thawed mouse pronuclear embryos in the presence of beta-mercaptoethanol in post-thaw culture.

The objective of this study was to investigate the effects of beta-mercaptoethanol (ß-ME) on post-thaw embryo developmental competence and implantation rate of mouse pronuclear (PN) embryos that were cryopreserved after slow freezing, solid surface vitrification (SSV) or open-pulled straw (OPS) vitrification methods. Mouse PN embryos were cryopreserved by using slow freezing, SSV and OPS methods. After cryopreservation, freeze-thawed PN embryos were cultured up to blastocyst stage in a defined medium supplemented without or with 50 µM ß-ME. The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (40.0%) or vitrified by OPS method (18.3%) were lower than those vitrified by SSV method (55.6%) and fresh embryos (61.9%) in the absence of 50 ß-ME in the culture media (p < 0.05). The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (53.1%) or by OPS method (41.9%) were lower than those vitrified by SSV method (79.5%) and that of fresh (85.7%) in the presence of ß-ME in the culture media (p < 0.05). The embryos transfer results revealed that the implantation rate of blastocyst derived from mouse PN embryos vitrified by SSV method (31.9% vs 51.2%) was similar to that of the control (39.0% vs 52.5%), but higher than those cryopreserved by slow freezing (28.2% vs 52.0%) and by OPS method (0.0% vs 51.2%) (p < 0.05). In conclusion, supplementation of ß-ME in an in vitro culture medium was shown to increase survival of embryo development and implantation rate of frozen-thawed mouse PN embryos after different cryopreservation protocols.

Efficient reproduction of cynomolgus monkey using pronuclear embryo transfer technique.

One of the technical bottlenecks in producing nonhuman primate models is that current assisted reproductive techniques, such as in vitro culture and frozen conservation of multicell-stage embryos, often result in poor embryo quality and subsequently lead to low birth rates. We investigated whether pronuclear embryo transfer can be used as an effective means for improving pregnancy and live birth rates of nonhuman primates. We collected 174 metaphase II oocytes by laparoscopy from 22 superovulated mature females and then fertilized these eggs using either in vitro fertilization or intracytoplasmic sperm injection, resulting in a 33.3% and a 50% fertilization rate, respectively. These 66 fertilized pronuclear-stage embryos were then tubally transferred to 30 recipients and led to 7 births and 1 abortion. Importantly, we observed that the highest live birth rate of approximately 64% was obtained when the transfer of pronuclear embryos was performed in the presence of new corpus luteum in the ovary of recipients between 24 h and 36 h after estradiol peak. Therefore, our experiments demonstrate that by matching the critical time window in the recipient's reproductive cycle for achieving optimal embryo-uterine synchrony, pronuclear embryo transfer technology can significantly improve the pregnancy rate and live birth of healthy baby monkeys. This efficient method should be valuable to the systematic efforts in construction of various transgenic primate disease models.

Pronuclear injection for the production of transgenic mice.

Transgenic mice have been instrumental in dissecting the role of various neuronal proteins under both physiological and pathological conditions. Pronuclear injection is the most widely used protocol for the generation of transgenic mice. Here, we describe all steps involved from DNA purification to the set up of a mouse colony including vasectomy, injection of the DNA into a donor zygote, transfer of injected zygotes into recipient foster mice, screening of offspring and establishment of transgenic mouse lines. We discuss the use of neuron-specific promoters to express proteins with a role in Alzheimer disease. Transgenic expression of a truncated form of the microtubule-associated protein tau (delta tau) is used as an example for the anticipated results.

Limited importance of pre-embryo pronuclear morphology (zygote score) in assisted reproduction outcome in the absence of embryo cryopreservation.

OBJECTIVE: To investigate the hypothesis that Z-score criteria represent a reliable predictor of implantation rate and pregnancy outcome in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles, excluding the possibility of embryo selection before the embryo transfer. DESIGN: Retrospective clinical study. SETTING: Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova (ASMN), Reggio Emilia, Italy. PATIENT(S): We analyzed 393 pregnancies obtained by IVF or ICSI cycles. INTERVENTION(S): Morphologic evaluations of Z-score in pre-embryos obtained from IVF or ICSI cycles. MAIN OUTCOME MEASURE(S): Evaluations of Z-scores, implantation rate, and clinical pregnancy outcome. RESULT(S): We did not find any statistically significant correlation between the Z-score of 1032 embryos transferred in 393 embryo transfers and the implantation rate or the pregnancy outcome. In particular, the best Z-score identified (Z1, 7.2%) did not seem to correlate with embryo implantation rate or pregnancy outcomes any better than those with worse scores (Z2, 6.9% and Z3, 85.9%). CONCLUSION(S): Our results seem to confirm that Z-score alone cannot be considered a better tool than standard morphologic criteria for identifying, controlling, or selecting embryos with a better chance of successful ongoing pregnancy.

Investigation and treatment of repeated implantation failure following IVF-ET.

Pregnancy rate following one cycle of IVF and ET can be as high as 60%. But even in the very successful units, some couples fail repeatedly. The causes for repeated implantation failure (RIF) may be because of reduced endometrial receptivity, embryonic defects or multifactorial causes. Various uterine pathologies, such as thin endometrium, altered expression of adhesive molecules and immunological factors, may decrease endometrial receptivity, whereas genetic abnormalities of the male or female, sperm defects, embryonic aneuploidy or zona hardening are among the embryonic reasons for failure of implantation. Endometriosis and hydrosalpinges may adversely influence both. In this mini review, we discuss the suggested methods for evaluation and treatment of RIF: repeated hysteroscopy, myomectomy, endometrial stimulation, immunotherapy, preimplantation genetic screening (PGS), assisted hatching, zygote intra-Fallopian transfer (ZIFT), co-culture, blastocyst transfer, cytoplasmic transfer, tailoring stimulation protocols and salpingectomy for hydrosalpinges.

Aneuploid analysis of tripronuclear zygotes derived from in vitro fertilization and intracytoplasmic sperm injection in humans.

This study demonstrates that the diploid ratio of tripronuclear zygotes after intracytoplasmic sperm injection (ICSI) is significantly higher as compared with that after conventional IVF; the extra pronucleus of tripronuclear zygotes after ICSI are mostly from the second polar body, not from sperm.

Number of transferred embryos: how to reduce multiple pregnancies.

Because the diagnostic tools for predicting whether an early cleavage stage embryo can lead to a viable pregnancy are still elusive, transfer of more than one embryo remains quite common. However, the only way to reduce multiple pregnancies, considered as the main adverse effect of assisted reproductive technology, is to transfer a single embryo. In countries such as Switzerland and Germany, the law allows cryopreservation only at the 2-pronuclear stage. This restricts considerably the possibility of selecting the embryos to be transferred. Therefore, a good cryopreservation program at the 2-pronuclear stage is an essential tool to optimize the efficiency of in vitro fertilization (IVF). We therefore recommend the Cumulated Singleton Delivery Rate (CUSIDERA) as a measure of standard IVF efficiency. This rate averages approximately 23.5% when calculated over the last 10 years in our unit and reaches a value above 35% for patients with more than 10 zygotes. Elective single-embryo transfers and the decrease of iatrogenic multiple pregnancies in IVF remain dependent on better prognostic tools for the appropriate selection of patients, gametes, and zygotes.