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.

XEN implant device versus trabeculectomy, either alone or in combination with phacoemulsification, in open-angle glaucoma patients.

PURPOSE: To compare the efficacy and safety of the XEN45 implant with that of trabeculectomy (TRAB), either alone or in combination with phacoemulsification (PHACO), in patients with open-angle glaucoma (OAG). METHODS: Retrospective, single-center and comparative study conducted on OAG patients. Patients were divided into four groups: group 1 (XEN alone); group 2 (XEN+PHACO); group 3 (TRAB alone); group 4 (TRAB+PHACO). For statistical purposes, groups 1 and 2 were combined (XEN implant), while groups 3 and 4 were also combined (TRAB surgery). The main outcome measure was intraocular pressure (IOP). RESULTS: Ninety-one patients (121 eyes; 65 XEN and 56 TRAB) were included. IOP reduction was - 6.7 (- 10.4 to - 3.0) mmHg, p = 0.0013; - 3.5 (- 5.0 to - 2.0) mmHg, p < 0.0001; - 8.1 (- 10.4 to - 5.9) mmHg, p < 0.0001l; and - 7.3 (- 9.3 to - 5.3) mmHg, p < 0.0001 in the XEN alone, XEN+PHACO, TRAB alone, and TRAB+PHACO, respectively. At month 12, an IOP ≥ 6 and ≤ 16 mm without treatment was achieved by 44 (67.7%) and 43 (76.8%), p = 0.2687 in the XEN implant and the TRAB surgery groups, respectively. The mean number of antiglaucoma medications was significantly reduced in all the study groups (p < 0.0001 each). Needling occurred in 20.0% (13/65) of eyes in the XEN implant group, while hyphema occurred in 30.4% (17/56) of eyes in the TRAB group. CONCLUSIONS: XEN implant, either alone or in combination with phacoemulsification, significantly reduces both IOP and the number of antiglaucoma medications to a similar rate than trabeculectomy, but with a better safety profile.

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.

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.

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.

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).

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.