Differential Gene Regulation of the Human Blastocyst Trophectoderm and Inner Cell Mass by Progesterone.

Knowledge of action of progesterone (P4) on the human preimplantation embryo is lacking. The objective of this study was to determine expression of a mitochondrial P4 receptor (PR-M) in the trophectoderm (TE) and the inner cell mass (ICM) of the human blastocyst and to determine P4-induced gene expression during growth from the cleavage to the blastocyst stage. Previously cryopreserved cleavage stage embryos were treated with P4 (10-6 M) or vehicle until blastocyst development. Cells from the TE and the ICM of dissected euploid embryos underwent RNA-seq analysis, while other embryos were used for analysis of nuclear PR (nPR) and PR-M expression.PR-M expression was confirmed in the TE, the ICM, and a human embryonic stem cell line (HESC). Conversely, nPR expression was absent in the TE and the ICM with low expression in the HESC line. RNA-seq analysis revealed P4 effects greater in the TE with 183 significant pathway changes compared to 27 in the ICM. The TE response included significant upregulation of genes associated with DNA replication, cell cycle phase transition and others, exemplified by a 7.6-fold increase in the cell proliferation gene, F-Box Associated Domain Containing. The majority of ICM pathways were downregulated including chromosome separation, centromere complex assembly and chromatin remodeling at centromere. This study confirms that human blastocysts express PR-M in both the TE and the ICM, but lack expression of nPR. P4-induced gene regulation differs greatly in the two cell fractions with the predominant effect of cell proliferation in the TE and not the ICM.

Association between donor gamete use and supernumerary embryo disposition decisions.

PURPOSE: The aim of this study is to determine if donor gamete use is associated with patients' decisions regarding disposition of supernumerary embryos. METHODS: Patients who intended to undergo an IVF cycle at a single academic center signed an embryo disposition consent form to indicate their disposition preferences for any supernumerary embryos. A retrospective chart review was performed to obtain the embryo disposition declarations and demographic information. The primary outcome was the distribution of embryo disposition choices between patients who used donor gametes compared to patients who did not use donor gametes. Fisher's exact test was used to compare groups. Logistic regression models were created to determine the association between donor gamete use and disposition decision after adjusting for patient age, body mass index, and nulliparity. RESULTS: Five hundred six patients were included. Ninety-one (18.0%) patients used donor gametes [46 (9.0%) donor oocytes, 52 (10.3%) donor sperm]. Patients using donor gametes differed from those not using donor gametes when making decisions concerning death of the patient (P < 0.01), simultaneous death (P = 0.04), separation (P < 0.01), discontinuation of ART (P = 0.01), and time-limited storage (P < 0.01). Most patients, regardless of donor or autologous gamete use, awarded embryos to themselves or their partner if given the option. For patients who did not choose this option, excess embryos were generally awarded to research or discarded rather than donating to another couple. Patients using donor gametes were more likely to award embryos to research over discarding. CONCLUSION: Patients using donor gametes made different choices regarding supernumerary embryo disposition compared to patients not using donor gametes.

Refraining from use diminishes cannabis-associated epigenetic changes in human sperm.

Cannabis use alters sperm DNA methylation, but the potential reversibility of these changes is unknown. Semen samples from cannabis users and non-user controls were collected at baseline and again following a 77-day period of cannabis abstinence (one spermatogenic cycle). Users and controls did not significantly differ by demographics or semen analyses. Whole-genome bisulfite sequencing identified 163 CpG sites with significantly different DNA methylation in sperm between groups (P < 2.94 × 10-9). Genes associated with altered CpG sites were enriched with those involved in development, including cardiogenesis and neurodevelopment. Many of the differences in sperm DNA methylation between groups were diminished after cannabis abstinence. These results indicate that sustained cannabis abstinence significantly reduces the number of sperm showing cannabis-associated alterations at genes important for early development.

Progesterone Increases Mitochondria Membrane Potential in Non-human Primate Oocytes and Embryos.

Mitochondrial activity is critical and correlates with embryo development. The identification of a novel human mitochondrial progesterone receptor (PR-M) that increases cellular respiration brings into question a role for progesterone in oocyte and preimplantation embryo development. Oocytes and embryos were generated from three Rhesus non-human primates (Macaca mulatta) undergoing in vitro fertilization. Immunohistochemical (IHC) staining for the progesterone receptor and mitochondria, RT-PCR with product sequencing for a mitochondrial progesterone receptor, and mitochondrial membrane determination with JC-1 staining were performed. IHC staining with selective antibodies to the progesterone receptor showed non-nuclear staining. Staining was absent in mouse control embryos. RT-PCR with product sequencing demonstrated PR-M transcript in Rhesus oocytes and embryos, which was absent in mouse embryos. Treatment of Rhesus oocytes and embryos with progesterone showed increased mitochondrial membrane potential, which was absent in mouse embryos. Our results support that progesterone increases mitochondrial membrane potential in oocytes and developing embryos. This is likely an in vivo mechanism to support preimplantation embryo development, and brings up the possibility of in vitro manipulation of culture media for optimization of growth.

Cannabinoid exposure and altered DNA methylation in rat and human sperm.

Little is known about the reproductive effects of paternal cannabis exposure. We evaluated associations between cannabis or tetrahydrocannabinol (THC) exposure and altered DNA methylation in sperm from humans and rats, respectively. DNA methylation, measured by reduced representation bisulfite sequencing, differed in the sperm of human users from non-users by at least 10% at 3,979 CpG sites. Pathway analyses indicated Hippo Signaling and Pathways in Cancer as enriched with altered genes (Bonferroni p < 0.02). These same two pathways were also enriched with genes having altered methylation in sperm from THC-exposed versus vehicle-exposed rats (p < 0.01). Data validity is supported by significant correlations between THC exposure levels in humans and methylation for 177 genes, and substantial overlap in THC target genes in rat sperm (this study) and genes previously reported as having altered methylation in the brain of rat offspring born to parents both exposed to THC during adolescence. In humans, cannabis use was also associated with significantly lower sperm concentration. Findings point to possible pre-conception paternal reproductive risks associated with cannabis use.

Freezing of all embryos in in vitro fertilization is beneficial in high responders, but not intermediate and low responders: an analysis of 82,935 cycles from the Society for Assisted Reproductive Technology registry.

OBJECTIVE: To assess in vitro fertilization (IVF) and pregnancy outcomes in patients having their first frozen embryo transfer (FET) after a freeze-all cycle versus similar patients having their first fresh embryo transfer (ET). DESIGN: Retrospective cohort study. SETTING: None. PATIENT(S): Registry data on 82,935 patient cycles from the Society for Assisted Reproductive Technology (SART). INTERVENTION(S): All first fresh autologous IVF cycles were analyzed and compared to first FET cycles after a freeze-all first IVF stimulation. The cycles were subdivided into cohorts based upon the number of oocytes retrieved (OR): 1-5 (low), 6-14 (intermediate), and 15+ (high responders). Univariate analyses were performed on cycle characteristics, and multivariable regression analyses were performed on outcome data. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (CPR) and live-birth rate (LBR). RESULTS: Of the 82,935 cycles analyzed, 69,102 patients had their first fresh transfer, and 13,833 had a first FET. High responders were found to have a higher CPR and LBR in the FET cycles compared with the fresh ET cycles (61.5 vs. 57.4%; 52.0 vs. 48.9%). In intermediate responders, both CPR and LBR were higher after fresh ET compared with FET (49.6% vs. 44.2%; 41.2 vs. 35.3%). Similarly, in low responders, CPR and LBR were higher after fresh compared with FET (33.2% vs. 15.9%; 25.9% vs. 11.5%). CONCLUSION(S): A freeze-all strategy is beneficial in high responders but not in intermediate or low responders, thus refuting the idea that freeze-all cycles are preferable for all patients.

Relationship between pre-embryo pronuclear morphology (zygote score) and standard day 2 or 3 embryo morphology with regard to assisted reproductive technique outcomes.

OBJECTIVE: To test the hypothesis that pregnancy rates are low if grade Z1 pre-embryos are not available for transfer and to determine if pronuclear morphology is a better predictor of pregnancy than traditional embryo morphology. DESIGN: Prospective clinical study. SETTING: Academic human reproduction laboratory. PATIENT(S): One hundred couples undergoing IVF with conventional insemination or ICSI. INTERVENTION(S): Embryo quality was assessed using both pre-embryo pronuclear morphology (zygote scoring or Z-scoring) at the time of fertilization evaluation and standard day 2 and day 3 embryo morphology (number of blastomeres and grading based on degree of fragmentation and blastomere size). MAIN OUTCOME MEASURE(S): We tested two decision models, one based on Z-scores and another on morphology, to determine which grading system better predicted pregnancy outcomes in assisted reproductive technique. Zygote score and embryo morphology were measured for all embryos and the transferred embryo pool. Implantation and pregnancy rates resulting from the embryo transfers of all cycles were calculated. RESULT(S): The Z-score distribution of 552 embryos was 27% Z1, 8% Z2, 50% Z3, and 15% Z4. Z1 and Z3 embryos had significantly (P approximately .03) higher quality over Z2 and Z4 embryos. Using the Z-score decision model with Z1 embryos having highest priority for transfer, pregnancy rates were similar between Z1 and Z3 embryos. Using embryo morphology as a decision model, pregnancy rates were highest in transfers containing one or two "best"-quality embryos. CONCLUSION(S): Z1 and Z3 embryos had similar morphology and pregnancy rates. The decision model based on the Z-score model was not better than standard embryo morphology in predicting pregnancy outcome.

Redefining the relationship between sperm deoxyribonucleic acid fragmentation as measured by the sperm chromatin structure assay and outcomes of assisted reproductive techniques.

OBJECTIVE: To test the hypothesis that couples with sperm chromatin structure assay (SCSA) DNA fragmentation index (DFI) values >27% would not achieve pregnancy with assisted reproductive techniques (ART) and to investigate how DFI and high DNA stainability (HDS), as measured by the SCSA, affect fertilization, cleavage, implantation, and pregnancy rates in IVF cycles. DESIGN: Prospective clinical study. SETTING: Academic human reproduction laboratory. PATIENT(S): One hundred couples undergoing IVF with conventional insemination or intracytoplasmic sperm injection. INTERVENTION(S): Testing with SCSA was performed by SCSA Diagnostics (Brookings, South Dakota) on a semen aliquot taken from ejaculate used for ART. MAIN OUTCOME MEASURE(S): Relating total DFI and HDS to conventional semen parameters and cycle-specific outcomes after ART. RESULT(S): Nine of nineteen couples achieved clinical pregnancy when DFI was > or =27%, and 2 of 22 couples achieved clinical pregnancy when DFI was < or =9%. One of nine couples achieved clinical pregnancy with HDS >17%. The DFI was negatively correlated with sperm density (r = -0.23, P<.03) and motility (r = -0.55, P<.00), and HDS was negatively correlated with sperm density (r = -0.37, P<.00). CONCLUSION(S): Sperm chromatin structure assay failed to identify elevated DFI thresholds for negative pregnancy outcome after ART. Patients with low DFI (< or =9%) were least likely to become pregnant, which is also contradictory to SCSA marketing, which states that DFIs of < or =15% have excellent fertility potential. Patients with HDS > or =17% had low pregnancy rates, indicating decreased fertility potential, which deserves further investigation. Larger studies are necessary to confirm that low DFI is associated with decreased fertility and, if proved, might redefine the use of the SCSA in evaluating infertility.