Comparative single-cell transcriptomic profiles of human androgenotes and parthenogenotes during early development.

OBJECTIVE: To unravel the differential transcriptomic behavior of human androgenotes (AGs) and parthenogenotes (PGs) throughout the first cell cycles, analyze the differential expression of genes related to key biologic processes, and determine the time frame for embryonic genome activation (EGA) in AGs and PGs. DESIGN: Laboratory study. SETTING: Private fertility clinic. PATIENT(S): Mature oocytes were retrieved from healthy donors and subjected to artificial oocyte activation using calcium ionophore and puromycin to generate PGs (n = 6) or enucleated and subjected to intracytoplasmic sperm injection to generate AGs (n = 10). INTERVENTION(S): Uniparental constructs at different early stages of development were disaggregated into constituent single cells (we suggest the terms parthenocytes and androcytes) to characterize the single-cell transcriptional landscape using next-generation sequencing. MAIN OUTCOMES MEASURE(S): Transcriptomic profiles comparison between different stages of early development in AGs and PGs. RESULT(S): The uniparental transcriptomic profiles at the first cell cycle showed 68 down-regulated and 26 up-regulated differentially expressed genes (DEGs) in PGs compared with AGs. During the third cell cycle, we found 60 up-regulated and 504 down-regulated DEGs in PGs compared with AGs. In the fourth cell cycle, 1,771 up-regulated and 1,171 down-regulated DEGs were found in PGs compared with AGs. The AGs and PGs had reduced EGA profiles during the first 3 cell cycles, and a spike of EGA at the fourth cell cycle was observed in PGs. CONCLUSION(S): Transcriptomic analysis of AGs and PGs revealed their complementary behavior until the fourth cell cycle. Androgenotes undergo a low wave of transcription during the first cell cycle, which reflects the paternal contribution to cell cycle coordination, mechanics of cell division, and novel transcription regulation. Maternal transcripts are most prominent in the third and fourth cell cycles, with amplification of transcription related to morphogenic progression and embryonic developmental competence acquisition. Regarding EGA, in PGs, a primitive EGA begins at the 1-cell stage and gradually progresses until the 4-cell stage, when crucial epigenetic reprogramming (through methylation) is up-regulated. In addition, our longitudinal single-cell transcriptomic analysis challenges that the zygote and early cleavage stages are the only totipotent entities, by revealing potential totipotency in cleavage-stage AGs and implications of paternal transcripts.

Rescuing monopronucleated-derived human blastocysts: a model to study chromosomal topography and fingerprinting.

OBJECTIVE: To quantify the percentage of monopronuclear-derived blastocysts (MNBs) that are potentially useful for reproductive purposes using classic and state-of-the-art chromosome analysis approaches, and to study chromosomal distribution in the inner cell mass (ICM) and trophectoderm (TE) for intertissue/intratissue concordance comparison. DESIGN: Prospective experimental study. SETTING: Single-center in vitro fertilization clinic and reproductive genetics laboratory. PATIENT(S): A total of 1,128 monopronuclear zygotes were obtained between June 2016 and December 2018. INTERVENTION(S): MNBs were whole-fixed or biopsied to obtain a portion of ICM and 2 TE portions (TE1 and TE2) and were subsequently analyzed by fluorescence in situ hybridization, new whole-genome sequencing, and fingerprinting by single-nucleotide polymorphism array-based techniques (a-SNP). MAIN OUTCOME MEASURE(S): We assessed MNB rate, ploidy rate, and chromosomal constitution by new whole-genome sequencing, and parental composition by comparative a-SNP, performed in a "trio"-format (embryo/parents). The 24-chromosome distribution was compared between the TE and the ICM and within the TE. RESULT(S): A total of 18.4% of monopronuclear zygotes progressed to blastocysts; 77.6% of MNBs were diploid; 20% of MNBs were male and euploid, which might be reproductively useful. Seventy-five percent of MNBs were biparental and half of them were euploid, indicating that 40% might be reproductively useful. Intratissue concordance (TE1/TE2) was established for 93.3% and 73.3% for chromosome matching. Intertissue concordance (TE/ICM) was established for 78.8%, but 57.6% for chromosome matching. When segmental aneuploidy was not considered, intratissue concordance and chromosome matching increased to 100% and 80%, respectively, and intertissue concordance and chromosome matching increased to 84.8% and 75.8%, respectively. CONCLUSION(S): The a-SNP-trio strategy provides information about ploidy, euploidy, and parental origin in a single biopsy. This approach enabled us to identify 40% of MNBs with reproductive potential, which can have a significant effect in the clinical setting. Additionally, segmental aneuploidy is relevant for mismatched preimplantation genetic testing of aneuploidies, both within and between MNB tissues. Repeat biopsy might clarify whether segmental aneuploidy is a prone genetic character.

Segmental aneuploidy in human blastocysts: a qualitative and quantitative overview.

BACKGROUND: Microarray-based and next generation sequencing (NGS) technologies have revealed that segmental aneuploidy is frequently present in human oocytes, cleavage-stage embryos and blastocysts. However, very little research has analyzed the type, size, chromosomal distribution and topography of the chromosomal segments at the different stages of development. METHODS: This is a retrospective study of 822 PGT-A (preimplantation genetic test for aneuploidies) performed on trophectoderm samples from 3565 blastocysts biopsied between January 2016 and April 2017. The cycles in question had been initiated for varying clinical indications. Samples were analyzed by next generation sequencing-based technology. Segmental aneuploidies were evaluated when fragment size was > 5 Mb. Blastocysts presenting a single segmental aneuploidy (SSA), without any additional whole-chromosome gain/loss, were statistically analyzed for incidence, type, size and chromosomal emplacement. Segment sizes relative to the whole chromosome or arm (chromosome- and arm-ratios) were also studied. RESULTS: 8.4% (299/3565) of blastocysts exhibited segmental aneuploidy for one or more chromosomes, some of which were associated with whole-chromosome aneuploidy while others were not. Nearly half of them (4.5%: 159/3565 of blastocysts) exhibited pure-SSA, meaning that a single chromosome was affected by a SSA. Segments were more frequent in medium-sized metacentric or submetacentric chromosomes and particularly in q-chrmosome arms, variables that were related to trophectoderm quality. SSA size was related to a greater extent to chromosome number and the arm affected than it was to SSA type. In absolute values (Mb), SSA size was larger in large chromosomes. However, the SSA:chromosome ratio was constant across all chromosomes and never exceeded 50% of the chromosome. CONCLUSIONS: SSA frequency is chromosome- and topographically dependent, and its incidence is not related to clinical or embryological factors, but rather to trophectoderm quality. SSA might be originated by chromosome instability in response to chromothripsis, bias introduced by the biopsy and/or iatrogenic effects. TRIAL REGISTRATION: Retrospectively registered.

Mitochondria as a tool for oocyte rejuvenation.

Ovarian aging leads to a decrease in the quantity and quality of oocytes. Aged oocytes have significantly reduced amounts of mitochondria, the energy factories of cells, leading to lower fertilization rates and poor embryonic development. Various techniques have tried to use heterologous or autologous sources of mitochondria to reestablish oocyte health by providing more energy. However, heterologous sources are no longer used owing to the known risk of heteroplasmy. Although autologous methods have recently been tested in humans, they have not shown a clear improvement in embryo quality. In this review, we describe the techniques that have been tested in recent years to provide a state of the art on oocyte rejuvenation through extra injection of mitochondria.

Autologous mitochondrial transfer as a complementary technique to intracytoplasmic sperm injection to improve embryo quality in patients undergoing in vitro fertilization-a randomized pilot study.

OBJECTIVE: To study if autologous mitochondrial transfer (AUGMENT) improves outcome in patients with previously failed in vitro fertilization (IVF). DESIGN: Randomized, controlled, triple-blind, experimental study. SETTING: Private infertility center, Valencian Institute of Infertility (IVI-RMA), Valencia, Spain. PATIENT(S): Infertile women ≤42 years of age, body mass index <30 kg/m2, antimüllerian hormone ≥4 pmol/L, >5 million/mL motile sperm, at least one previous IVF with at least five metaphase oocytes (MIIs) collected, and low embryo quality. INTERVENTIONS(S): An ovarian cortex biopsy was performed to isolate egg precursor cells to obtain their mitochondria. Sibling MIIs were randomly allocated to AUGMENT (experimental) or intracytoplasmic sperm injection (Control). In AUGMENT, mitochondrial suspension was injected along with the sperm. Viable blastocysts from both groups were biopsied for preimplantation genetic testing for aneuploidy. MAIN OUTCOME MEASURE(S): Pregnancy, embryo quality. RESULT(S): An interim analysis was conducted. The patients' mean age was 36.3 ± 3.6 years, and they had an average of 2.5 ± 1.5 previous IVF cycles. Two of the 59 enrolled patients spontaneously conceived (one miscarried). Fifty-seven patients had ovarian biopsies and underwent stimulation. Oocyte retrieval was performed in 56 patients (premature ovulation; n = 1). A total of 253 MIIs were inseminated in AUGMENT and 250 in Control; fertilization rates were 62.7 ± 30.0% and 68.7 ± 29.1%, respectively. Statistical differences were observed in day 5 blastocyst formation rates (23.3 ± 32.0% vs. 41.1 ± 36.9%). Neither the euploid rate per biopsied blastocyst (43.8 ± 41.7% vs. 63.8 ± 44.1%) nor the euploid rate per MII (9.8 ± 20.5% vs. 11.9 ± 16.1%) between AUGMENT and Control achieved statistical significance. Moreover, no differences were seen regarding mitochondrial DNA content and relevant morphokinetic variables. Thirty patients were able to undergo embryo transfer. Cumulative live birth rates per transferred embryo were 41.6% in AUGMENT and 41.2% in Control. CONCLUSION(S): AUGMENT does not seem to improve prognosis in this population. Therefore, the study has been discontinued. CLINICAL TRIAL REGISTRATION NUMBER: NCT02586298.

Kinetics of the early development of uniparental human haploid embryos.

OBJECTIVE: To describe morphokinetically the early development of human haploid parthenotes and androgenotes and to compare them with euploid embryos. DESIGN: Experimental study of kinetics. SETTING: University-affiliated private fertility center. PATIENT(S): Experimental haploid parthenotes and androgenotes. INTERVENTION(S): Kinetic study of early development (up to eight cells) of 8 parthenotes, 10 androgenotes, and 20 euploid embryos. MAIN OUTCOME MEASURE(S): Timing of the first seven cleavages determined according to embryo origin, then calculation of the duration of the second and third cell cycles (cc2 and cc3) of whole embryos and individual cells. RESULT(S): Parthenotes and androgenotes were experimentally produced by artificial oocyte activation and intracytoplasmic sperm injection of enucleated oocytes, respectively. Uniparental embryos having 6 to 10 cells were assessed for haploidy, their kinetics analyzed, retrospectively compared with euploid embryos. All the first seven cleavages occurred later in parthenotes than in both androgenotes and correctly fertilized embryos. The whole embryos and single cells showed that cc2 was longer in parthenotes than in both androgenotes and correctly fertilized embryos; cc3 was shorter in androgenotes than in both parthenotes and correctly fertilized embryos. The duration of cc2 versus cc3 was longer in correctly fertilized embryos and parthenotes than in androgenotes. CONCLUSION(S): Parthenotes and androgenotes have different kinetics. The former have a longer cc2, and the latter a consistently shorter cc3 in comparison with correctly fertilized embryos.

Morphokinetic analysis and embryonic prediction for blastocyst formation through an integrated time-lapse system.

OBJECTIVE: To describe the events associated with the blastocyst formation and implantation that occur in embryos during preimplantation development based on the largest sample size ever described with time-lapse monitoring. DESIGN: Observational, retrospective, single-center clinical study. SETTING: University-affiliated private IVF center. PATIENT(S): A total of 7,483 zygotes from 990 first treatments of intracytoplasmic sperm injection (ICSI; 627 of oocyte donor vs. 363 autologous oocyte cycles), of which 832 blastocysts were transferred. INTERVENTION(S): No patient intervention. Embryos were cultured in a time-lapse monitoring system, and the embryos were transferred on day 5 after ICSI. Embryo selection was based on the multivariable model previously developed and on blastocyst morphology. MAIN OUTCOME MEASURE(S): Using a time-lapse system, embryo images were acquired every 15 minutes for 120 hours. Embryos cleavage time points up to the 9-cell stage (t2-t9) as well as to the morula stage (tM) and blastocyst formation (tB) were registered in hours after ICSI. Additionally, duration of the cell cycle and synchrony of the second and third cell cycles were defined. As a result, we have monitored the embryonic development of a total of 3,215 blastocysts, of which 832 were transferred. Finally, we analyzed the characteristics of embryonic development of blastocyst (phase 1) and of implanted and not implanted (phase 2) embryos as finally validated in an independent data set (phase 3). RESULT(S): A detailed retrospective analysis of cleavage times was made for 7,483 zygotes. We analyzed 17 parameters and found several significantly correlated with subsequent blastocyst formation and implantation. The most predictive parameters for blastocyst formation were time of morula formation, tM (81.28-96.0 hours after ICSI), and t8-t5 (≤8.78 hours) or time of transition of 5-blastomere embryos to 8-blastomere embryos with a receiver operating characteristic curve (ROC) value = 0.849 (95% confidence interval [CI], 0.835-0.854; phase 1). These parameters were less predictive of implantation, with a ROC value = 0.546 (95% CI, 0.507-0.585). We also observed that time for expansion blastocyst, tEB (107.9-112.9 hours after ICSI), and t8-t5 (≤5.67 hours after ICSI) predict blastocyst implantation, with a ROC value = 0.591 (95% CI, 0.552-0.630; phase 2). The model was validated on an independent data set and gave a ROC of 0.596 (0.526-0.666; phase 3). CONCLUSION(S): The inclusion of kinetic parameters into score evaluation may improve blastocyst selection criteria and can predict blastocyst formation with high accuracy. We propose two multivariable models based on our findings to classify embryos according to their probabilities of blastocyst stage and implantation in the largest data set ever reported of human blastocysts.

Morphokinetics as a predictor of self-correction to diploidy in tripronucleated intracytoplasmic sperm injection-derived human embryos.

OBJECTIVE: To describe, in morphokinetic terms, a tripronucleated embryo (TPN) population according to ploidy and to explore the value of such variables for predicting ploidy. DESIGN: Experimental. SETTING: In vitro fertilization laboratory. PATIENT(S): Seventy-nine TPN embryos obtained after intracytoplasmic sperm injection (TPN-ICSI) were cultured in a time-lapse incubator for 6 days. INTERVENTION(S): Ploidy determinations were carried out for 35 TPN-ICSI at the cleavage and/or blastocyst stage. Their morphokinetics were then retrospectively compared. MAIN OUTCOME MEASURE(S): Direct (cleavage time from 2- to 8-cell stages) and indirect (cell cycle duration and blastomere synchrony at cleavage) morphokinetic variables; ploidy determination by FISH; in vitro development to the blastocyst stage. RESULT(S): TPN-ICSI cleaved later than bipronucleated control embryos (BPN). Diploid TPN displayed morphokinetic behavior closer to BPN than triploid TPN regarding almost all of the direct and indirect morphokinetic variables measured. Variable t5 was found to be a predictable variable of ploidy in TPN. CONCLUSION(S): TPN-ICSI are not homogeneous in ploidy, cleavage, or morphokinetic terms. Diploid, but nontriploid, TPN are morphokinetically similar to diploid BPN. The ploidy of TPN can be predicted by variable t5.

The human first cell cycle: impact on implantation.

The morphology of fertilization events has been related to successful implantation by subjective criteria (pronuclei score, pronuclei symmetry and position). This work first described these events by time-lapse technology and then compared the timings of fertilization events (second polar body extrusion, first and second pronuclei appearance, abuttal and fading) in implanted versus nonimplanted embryos in a 2-year cohort retrospective study. A total of 1448 transferred embryos from 842 patients undergoing intracytoplasmic sperm injection with oocyte donation were monitored, 212 embryos from treatments where the number of gestational sacs matched the number of transferred embryos and 687 embryos from treatments no biochemical pregnancy was achieved. The timings at which second polar body extrusion (3.3-10.6 h), pronuclear fading (22.2-25.9 h) and length of S-phase (5.7-13.8 h) occurred were linked successfully to embryo implantation. The other parameters were apparently not related, as determined by image acquisition and time-lapse analysis.

Short-term storage of tripronucleated human embryos.

PURPOSE: To determine the survival and subsequent in vitro development of human cleavage stage embryos and hatched blastocysts following varying periods of short-term storage at 4 °C, using tripronucleated human embryos (TPN) as a model. METHODS: TPN cleavage embryos and hatched blastocysts short-term stored at 4 °C for 0 h (control), 24 h and 48 h. The main outcome measures were: survival rates (SR) and in vitro developmental ability (blastocyst rate and blastocyst-re-expansion rate) in each of the groups after storage. RESULTS: Cleavage-stage TPN survived at comparable rates to controls, regardless of storage time (average: 97.3 %). The in vitro development of cleavage-stage TPN stored for 24 h was comparable to that of controls (average 64.7 %), but was significantly impaired when storage lasted 48-h (20.8 %). After artificial shrinkage, SR was comparable in 24-h-stored and non-stored hatched blastocysts (85.7 %; p > 0.05), but was significantly impaired in the 48-h-stored group (20.0 %). Following 24-h storage, the re-expansion rate of hatched blastocysts was similar to that of controls (average: 57.1 %; p > 0.05), but was higher than that of the 48-h-stored group (15.0 %; p < 0.05). CONCLUSIONS: TPN human cleavage embryos and blastocysts can be successfully stored short-term for up to 24 h at 4 °C without using cryoprotectants without any significant negative impact on survival or subsequent in vitro development.

Spontaneous in vitro maturation of oocytes prior to ovarian tissue cryopreservation in natural cycles of oncologic patients.

PURPOSE: To evaluate the recovery rate and spontaneous in vitro maturation (IVM) of immature oocytes enclosed within or released from follicles during the processing of ovarian tissue prior to its cryopreservation. METHODS: Thirty-three oncologic patients who had not previously undergone chemo or radiotherapy underwent ovarian tissue cryopreservation (OTC) during natural menstrual cycles. Immature oocytes, enclosed within follicles or released during ovarian cortex processing, were collected and matured spontaneously in vitro for 48 h. Nuclear maturation was assessed every 24 h and the ability of the IVM oocytes to display a normal activation response following parthenogenetic activation was evaluated. The following outcome measures were also evaluated: disease, age, FSH, LH, E2, P4 and AMH serum levels, menstrual cycle day, recovery and spontaneous IVM and parthenogenetic activation rates. RESULTS: Oocytes recovered per patient were 3.3 ± 0.7 (1.8-4.7 oocytes, 95CI), regardless of the menstrual phase. The mean number of IVM oocytes per patient was 1.3 ± 0.2 oocytes (95CI: 0.8-1.8), regardless of menstrual phase (p = 0.86) and oocyte origin (p = 0.61). Forty-one percent of oocytes extruded the second polar body and formed one pronucleus after parthenogenetic activation. CONCLUSION: Twenty-one of the 33 women (63.6 %) requesting OTC produced at least one mature oocyte.

Limited implantation success of direct-cleaved human zygotes: a time-lapse study.

OBJECTIVE: To evaluate embryos with direct cleavage (≤5 hours) from two to three cells (DC2-3) and correlate this morphokinetic parameter to implantation and ongoing pregnancy. DESIGN: Clinical multicenter retrospective study. SETTING: Private in vitro fertilization (IVF) centers. PATIENT(S): From three clinics, a total of 979 treatments including 5,225 embryos using autologous or donated oocytes, of which 1,659 embryos were transferred. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical pregnancy confirmed by ultrasound in week 7. RESULT(S): Of the total embryo cohort, 715 (13.7%) underwent direct cleavage from two to three cells, 1,659 embryos were transferred to recipients, and 109 of the transferred embryos cleaved directly from two to three cells (6.6%). Only one DC2-3 embryo was known to result in a clinical pregnancy (1%) and 80 (73.4%) DC2-3 embryos did not implant. Of the 1,550 embryos transferred not showing DC2-3, 203 embryos were from treatments with 100% implantation (13.1%), and 804 (51.8%) embryos did not implant. The known implantation rate of DC2-3 embryos was statistically significantly lower than for embryos with a normal cleavage pattern (1.2% vs. 20.2%, respectively). CONCLUSION(S): Embryos with DC2-3 had a statistically significantly lower implantation rate than embryos with a normal cleavage pattern, suggesting that rejection of these embryos for transfer could improve the implantation rate.

The dynamics of in vitro maturation of germinal vesicle oocytes.

OBJECTIVE: To evaluate the dynamics of the nuclear maturation (NM) of in vitro-matured (IVM) oocytes and to determine the most favorable duration of meiosis II (MII) arrest in relation to the normal activation response. DESIGN: Experimental. SETTING: University-affiliated infertility clinic. PATIENT(S): Donated immature germinal vesicle oocytes (GV). INTERVENTION(S): The GV underwent spontaneous IVM and the dynamics of NM studied by real-time monitoring. The IVM oocytes were parthenogenetically activated at different MII arrest points and their response assessed. MAIN OUTCOME MEASURE(S): Moment of GV breakdown; extrusion of the first polar body; duration of MI and MII arrest; activation rate (AR) and type. RESULT(S): Two GV populations-early (E-IVM, 18.4 ± 2.7 hours) and late (L-IVM, 26.3 ± 3.8 hours) maturing-were defined according to the time required for extrusion of the first polar body. Significantly more E-IVM than L-IVM exhibited a normal activation response (61.3% vs. 34.6%), but AR were similar (average, 88.6%) in both groups. Duration of the GV stage differed between the two groups, but MI arrest (14.0 ± 0.3 hours) was constant. The E-IVM arrested at MII for at least 4.3 hours displayed significantly lower AR and similar normal activation rates (61.3%) to E-IVM arrested for a shorter time (83.9% vs. 100%). The L-IVM displayed a similar AR (80.8%), but lower normal activation rates than E-IVM (34.6%), regardless of when activation took place. CONCLUSION(S): The success of IVM depends on the NM timing rather than on the length of MII arrest.

Self-correction in tripronucleated human embryos.

OBJECTIVE: To explore the occurrence of ploidy and parental self-correction in tripronuclear (TPN) human embryos. DESIGN: Experimental. SETTING: Research facility. PATIENT(S): Thirty-two TPN embryos resulting from intracytoplasmic sperm injection (ICSI-TPN) and 18 TPN embryos resulting from conventional IVF (IVF-TPN). INTERVENTION(S): Tripronuclear embryos were cultured in vitro for 6 days. Those with ≥ 6 cells were biopsied for ploidy analysis. Blastocysts were studied for ploidy or parental inheritance. Heteroparental inheritance was determined after comparing polymorphic loci in the genomic DNA of a blastocyst and in the parents' DNA. MAIN OUTCOME MEASURE(S): Tripronuclear origin, cell number at biopsy, chromosome analysis using fluorescent in situ hybridization, parental inheritance analysis using polymerase chain reaction amplification and sequencing, in vitro development to the blastocyst stage, and percentage of diploid and triploid cleavage embryos and blastocysts. RESULT(S): Half of ICSI-TPN embryos became self-corrected blastocysts whereas only one IVF-TPN embryo did. CONCLUSION(S): Both ICSI-TPN and IVF-TPN embryos are capable of self-correction, but the latter to a lesser extent. Neither parental inheritance nor ploidy determines the ability of a TPN embryo to progress to the blastocyst stage. However, the ability of a TPN embryo to become self-corrected is determined by the parental origin of the extra pronucleus.

Spontaneous in vitro maturation and artificial activation of human germinal vesicle oocytes recovered from stimulated cycles.

PURPOSE: To define germinal vesicles (GV) by morphometric and morphologic examination and by chromatin compaction and to assess their spontaneous nuclear and cytoplasmic competence. MATERIALS: 131 GV were cultured for 42.7 ± 2.4 h. Nuclear maturation was evaluated at four time points. Sixty-seven in vitro and twenty-five in vivo metaphase II (MII) were activated. Parthenotes with 2 PB and one pronucleus (NA) were studied for ploidy. RESULTS: A total of 74.8% GV matured to MII: 55% at 21.4 ± 2.4 h and 47.3% in the following 24 h. Artificial activation induced NA in 79.2% of in vivo-MII and in 22.4% of in vitro-MII. All NA were haploid. CONCLUSIONS: GV spontaneously mature at the nuclear level. Their NA are haploid, but their cytoplasmic competence is compromised. Variables were not found to be predictors of oocyte competence, probably due to our population being homogeneous with respect to most of the variables studied.

Vitrification of isolated human blastomeres.

This article describes a new methodology for preserving and banking isolated human blastomeres, whose originality is based on packing the blastomere into an emptied zona pellucida before vitrification. After warming, 75.7% of blastomeres survived and developed at a rate comparable to that in noncryopreserved blastomeres (62.5% cleavage, 26.6% compaction, and 20.3% cavitation).

Twins born after transplantation of ovarian cortical tissue and oocyte vitrification.

OBJECTIVE: To present a combination of ovarian tissue and oocyte cryopreservation as an effective strategy for achieving pregnancy in a breast cancer patient. DESIGN: Case report. SETTING: Tertiary care university-affiliated hospital, tissue bank, and infertility clinic. PATIENT(S): A 36-year-old patient diagnosed with atypical medullar breast cancer and negative for estrogen, P, and HER2 receptors underwent ovarian tissue cryopreservation before receiving chemotherapy and radiotherapy. INTERVENTION(S): Laparoscopic ovarian cortex extraction, ovarian tissue cryopreservation, ovarian tissue thawing and transplantation, controlled ovarian stimulation (COS), oocyte retrieval, vitrification and IVF, and embryo culture and replacement. MAIN OUTCOME MEASURE(S): Resumption of spontaneous ovarian function after transplantation, response to COS, oocyte vitrification, IVF, pregnancy, and delivery. RESULT(S): Menses occurred 63 days after transplantation. Sixteen mature oocytes were obtained in four COS procedures. All vitrified oocytes survived warming, and 77.7% were fertilized. Two day 3 embryos were replaced, and two healthy boys were born at 34 weeks. CONCLUSION(S): Ovarian tissue cryopreservation and grafting preserves fertility. Simultaneous oocyte vitrification increases the success of assisted reproductive technology in poor-prognosis patients and avoids the consequences of the short lifespan of the transplanted tissue.

Morphologic indicators predict the stage of chromatin condensation of human germinal vesicle oocytes recovered from stimulated cycles.

OBJECTIVE: To assess germinal vesicles (GV) recovered from stimulated cycles by means of morphometric and morphologic examination (using contrast-phase and image analysis) and chromatin configuration (using fluorescent DNA imaging), and to evaluate the relevance of morphometric and morphologic parameters as forecasters of chromatin status. DESIGN: Experimental study. SETTING: University-affiliated infertility clinic. PATIENT(S): One hundred and thirty-one GV oocytes donated to patients for intracytoplasmic sperm injection. INTERVENTION(S): We evaluated 131 GVs by means of morphology and morphometry with the use of contrast phase microscopy. They were subsequently fixed, DNA stained, and assessed by fluorescent microscopy. Compiled data were retrospectively grouped according to three models. MAIN OUTCOME MEASURE(S): Model A: ova were grouped according to chromatin condensation (noncondensed vs. condensed). Model B: ova were grouped according to chromatin distribution in relation to the nucleolus-like body (NLB) (not surrounding vs. surrounding and/or absent) but regardless of the condensation stage. Model C: GV oocytes were grouped according to the combination of both of the previously mentioned parameters (chromatin condensation and distribution in relation to the NLB). RESULT(S): According to the GV classification of model A, nucleoplasm, nucleus position, nuclear envelope continuity, and oocyte size were shown to be relevant and were included in a mathematical model for predicting chromatin condensation stage. CONCLUSION(S): Noninvasive analysis of GV oocytes using contrast-phase microscopy maintains oocytes in a viable state and allows the chromatin condensation status to be predicted.

Vitrification of preimplantation genetically diagnosed human blastocysts and its contribution to the cumulative ongoing pregnancy rate per cycle by using a closed device.

OBJECTIVE: To evaluate the survival rate and clinical results of our vitrification procedure on preimplantation genetic diagnosis (PGD) blastocysts and to calculate its actual contribution to the reproductive outcome per cycle. DESIGN: Retrospective clinical study. SETTING: University Institute IVI, Valencia, Spain. PATIENT(S): Patients who requested cryotransfer of surplus PGD blastocysts after failed fresh elective transfer. INTERVENTION(S): Retrospectively collected data during 2 years of experience with blastocyst vitrification. MAIN OUTCOME MEASURE(S): Primary outcome measures were the following: blastocyst recovery and survival; cryotransfer cancellation; and the implantation, pregnancy (PR), and ongoing-pregnancy rates. The secondary outcome measure was cumulative ongoing PR (COPR). RESULT(S): Cocultured vitrified PGD blastocysts were recovered and progressed in development after overnight culture (survival rate) at rates comparable to those of non-PGD blastocysts (49% and 42%, respectively). After transfer to 64% of patients, no statistical differences were found between PGD and non-PGD blastocyst groups concerning the following: PR (44% vs. 37%), implantation rate (40% vs. 27%), and ongoing-pregnancy rate (32% vs. 37%). Moreover, blastocyst vitrification significantly increased the COPR in both PGD and non-PGD cycles, from 47% (62/133) to 53% (70/133) and from 45% (24/53) to 53% (28/53), respectively. CONCLUSION(S): A preimplantation genetic diagnosis blastocyst vitrification procedure showed survival rates and improvements on the COPR that were comparable to those in non-PGD blastocyst cycles. Moreover, vitrification of biopsied and diagnosed embryos at the more advanced stages instead of at earlier cleavage stages is presented as an attractive strategy to consider in PGD programs.

Heteroparental blastocyst production from microsurgically corrected tripronucleated human embryos.

OBJECTIVE: To prove the efficiency of identification and removal of one of the surplus paternal pronuclei in dispermic IVF zygotes to obtain heteroparental blastocysts. DESIGN: Experimental. SETTING: One hundred fourteen tripronucleated (3PN) embryos from conventional IVF. PATIENT(S): After informed and signed consent, the patients from Instituto Valenciano Infertilidad (IVI), Valencia, donated their abnormally fertilized embryos. INTERVENTION(S): Seventy-two embryos were diploidized by microsurgical removal of the pronucleus located at the farthest position to the second polar body. Forty-two 3PN embryos served as controls. MAIN OUTCOME MEASURE(S): Survival and correction rate; in vitro development up to the blastocyst stage; X, Y, and 18 chromosome determination by triple fluorescent in situ hybridization and, inheritance analysis for 10 polymorphic repeat regions using polymerase chain reaction (PCR) amplification and sequencing. RESULT(S): Seventy-eight percent of 3PN zygotes (56/72) survived manipulation and eventually 51 zygotes had two pronuclei (71%). Forty-one percent of manipulated embryos progressed in vitro to the blastocyst stage (21/51). Fluorescent in situ hybridization analysis performed on eight manipulated embryos confirmed their diploid state; all four controls were triploid. Heteroparental inheritances were also confirmed in four of six manipulated embryos. CONCLUSION(S): Heteroparental blastocysts can be derived from corrected dispermic zygotes.