Are tri-pronuclear embryos that show two normal-sized pronuclei and additional smaller pronuclei useful for embryo transfer?

Purpose: This study aimed to analyze whether tripronuclear (3PN) zygotes, with two normal-sized PNs and an additional smaller PN (2.1PN), can be used for embryo transfer. Methods: A retrospective embryo cohort study was conducted on 695 patients who underwent intracytoplasmic sperm injection treatment. Blastocyst formation rates were compared between 2.1PN and 2PN zygotes and PGT-A analysis was performed on 15 blastocysts derived from 2.1PN zygotes. Results: Blastocyst formation rates were comparable between 2.1PN (43.8%) and 2PN zygotes (54.8%; p = 0.212). The rates of blastocysts with good morphology derived from 2.1 PN and 2PN zygotes were 18.8% and 25.5%, respectively. No significant differences were detected (p = 0.383). All of the analyzed blastocysts were diploid; however, 13 of these were found to be aneuploid, with a further two being mosaic. Conclusion: Our results suggest that 2.1PN embryos can reach blastocyst stage. These blastocysts were diploid, however, predominantly aneuploid, and therefore could not be used for embryo transfer.

Clinical outcomes of microdissection testicular sperm extraction and intracytoplasmic sperm injection in Japanese men with Y chromosome microdeletions.

PURPOSE: We investigated the clinical results of Japanese men with Y chromosome microdeletions. METHODS: This study retrospectively examined 2163 azoospermic or severe oligozoospermic patients. We investigated the frequency of azoospermia factor (AZF) deletions and sperm retrieval rate (SRR) by microTESE in patients with these deletions, then analyzed the ICSI outcomes. RESULTS: Azoospermia factor deletions were found in 201 patients. SRR was significantly higher than that of the control group (74.0% vs 20.4%, P < .001). Thirty-three couples underwent ICSI using testicular spermatozoa retrieved by microTESE, and eight couples underwent ICSI using ejaculatory spermatozoa. The fertilization rate and clinical pregnancy rate per embryo transfer cycle were significantly higher in the ejaculatory group than that of the testicular group (66.4% vs 43.7%, P < .001, 53.3% vs 24.7%, P = .03, respectively). When compared with the control group, the fertilization rate was significantly lower in the testicular group with AZFc microdeletions (43.7% vs 53.6%, P < .001). CONCLUSIONS: Our study highlights that although microTESE in azoospermic men with AZFc microdeletions led to a higher SRR, ICSI outcomes of these men were worse than that of men without AZF deletions, even if testicular spermatozoa were retrieved.

Clinical outcomes of MII oocytes with refractile bodies in patients undergoing ICSI and single frozen embryo transfer.

PURPOSE: This study aimed to analyze whether the presence of refractile bodies (RFs) negatively affects fertilization, embryo development, and/or implantation rates following intracytoplasmic sperm injection (ICSI). METHODS: This retrospective embryo cohort study involved a total of 272 patients undergoing ICSI treatment of blastocyst cryopreservation. RESULTS: In the study, no significant differences were found regarding 2PN formation rates between RF(+) (76.5%) and RF(-) oocytes (77.2%). However, the blastocyst formation rate on Day 5 in RF(+) oocytes was 45.8%, which was significantly lower than that of 52.2% in RF(-) oocytes (aOR 0.74, 95% CI 0.59-0.93, P = .011). Implantation rates were also significantly lower in RF(+) oocytes (24.2%) as compared to RF(-) oocytes (42.2%) (aOR 0.46, 95% CI 0.26-0.78, P = .005). Furthermore, the implantation rate of RF(+) oocytes (28.6%), when high-quality blastocysts were transferred, was significantly lower than that of RF(-) oocytes (46.1%) (aOR 0.50, 95% CI 0.25-0.96, P = .043). CONCLUSION: Our results suggest that oocytes with the presence of RFs have a lower potential for blastocyst development. Even when they develop into high-quality blastocysts, the chances of implantation are reduced.

Feasibility of predicting live birth by combining conventional embryo evaluation with artificial intelligence applied to a blastocyst image in patients classified by age.

PURPOSE: To identify the multivariate logistic regression in a combination (combination method) involving artificial intelligence (AI) classifiers in images of blastocysts along with a conventional embryo evaluation (CEE) to predict the probability of accomplishing a live birth in patients classified by maternal age. METHODS: Retrospectively, a total of 5691 blastocysts were enrolled. Images captured 115 hours or 139 hours if not yet sufficiently large after insemination were classified according to age as follows: <35, 35-37, 38-39, 40-41, and ≥42 years old. The classifiers for each category were created by using convolutional neural networks associated with deep learning. Next, the feasibility of a method combining AI with multivariate logistic model functions by CEE was investigated. RESULTS: The values of the area under the curve (AUC) and the accuracies to predict live birth achieved by the CEE/AI/combination methods were 0.651/0.634/0.655, 0.697/0.688/0.723, 0.771/0.728/0.791, 0.788/0.743/0.806 and 0.820/0.837/0.888, and 0.631/0.647/0.616, 0.687/0.675/0.671, 0.725/0.697/0.732, 0.714/0.776/0.801, and 0.910/0.866/0.784 for age categories of <35, 35-37, 38-39, 40-41, and ≥42 years old, respectively. CONCLUSIONS: Though there were mostly no significant differences regarding the AUC and the sensitivity plus specificity in all age categories, the combination method seemed to be the best.

A higher incidence of smooth endoplasmic reticulum clusters with aromatase inhibitors.

PURPOSE: This study aimed to analyze whether a regimen of aromatase inhibitor (AI) could reduce the occurrence of smooth endoplasmic reticulum clusters (sERCs) in oocytes. METHODS: The AI and the clomiphene citrate (CC) regimens were compared, regarding the sERC (+) rates and the serum estradiol and progesterone levels on the date of hCG administration, and the duration of AI, CC, and hMG administration. RESULTS: The occurrence of sERCs in oocytes from patients treated with AI was significantly higher than that in oocytes from those treated with CC. Both the serum estradiol and progesterone levels were found to be significantly higher in sERC (+) than in sERC (-) cycles. With regard to the CC cycles, no significant differences were detected. The duration of AI and hMG administration was longer for sERC (+) than for sERC (-) cycles. CONCLUSION: As AI did not reduce the occurrence of sERCs, the elevation of estradiol may not be the cause of sERC occurrence but a consequence. Considering the higher levels of progesterone and longer duration of hMG in sERC (+) cycles, the negative effects of premature luteinization, which frequently occur with the AI protocol, should be investigated further.

Feasibility of artificial intelligence for predicting live birth without aneuploidy from a blastocyst image.

PURPOSE: To make the artificial intelligence (AI) classifiers of the image of the blastocyst implanted later in order to predict the probability of achieving live birth. METHODS: A system for using the machine learning approaches, which are logistic regression, naive Bayes, nearest neighbors, random forest, neural network, and support vector machine, of artificial intelligence to predict the probability of live birth from a blastocyst image was developed. Eighty images of blastocysts that led to live births and 80 images of blastocysts that led to aneuploid miscarriages were used to create an AI-based method with 5-fold cross-validation retrospectively for classifying embryos. RESULTS: The logistic regression method showed the best results. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value were 0.65, 0.60, 0.70, 0.67, and 0.64, respectively. Area under the curve was 0.65 ± 0.04 (mean ± SE). Estimated probability of belonging to the live birth category was found significantly related to the probability of live birth (P < 0.005). CONCLUSIONS: Classifiers using artificial intelligence applied toward a blastocyst image have a potential to show the probability of live birth being the outcome.

Feasibility of deep learning for predicting live birth from a blastocyst image in patients classified by age.

PURPOSE: To identify artificial intelligence (AI) classifiers in images of blastocysts to predict the probability of achieving a live birth in patients classified by age. Results are compared to those obtained by conventional embryo (CE) evaluation. METHODS: A total of 5691 blastocysts were retrospectively enrolled. Images captured 115 hours after insemination (or 139 hours if not yet large enough) were classified according to maternal age as follows: <35, 35-37, 38-39, 40-41, and ≥42 years. The classifiers for each category and a classifier for all ages were related to convolutional neural networks associated with deep learning. Then, the live birth functions predicted by the AI and the multivariate logistic model functions predicted by CE were tested. The feasibility of the AI was investigated. RESULTS: The accuracies of AI/CE for predicting live birth were 0.64/0.61, 0.71/0.70, 0.78/0.77, 0.81/0.83, 0.88/0.94, and 0.72/0.74 for the age categories <35, 35-37, 38-39, 40-41, and ≥42 years and all ages, respectively. The sum value of the sensitivity and specificity revealed that AI performed better than CE (P = 0.01). CONCLUSIONS: AI classifiers categorized by age can predict the probability of live birth from an image of the blastocyst and produced better results than were achieved using CE.

Improved development of mouse and human embryos using a tilting embryo culture system.

Mammalian embryos experience not only hormonal but also mechanical stimuli, such as shear stress, compression and friction force in the Fallopian tube before nidation. In order to apply mechanical stimuli to embryos in a conventional IVF culture system, the tilting embryo culture system (TECS) was developed. The observed embryo images from the TECS suggest that the velocities and shear stresses of TECS embryos are similar to those experienced in the oviduct. Use of TECS enhanced the development rate to the blastocyst stage and significantly increased the cell number of mouse blastocysts (P<0.05). Although not statistically significant, human thawed embryos showed slight improvement in development to the blastocyst stage following culture in TECS compared with static controls. Rates of blastocyst formation following culture in TECS were significantly improved in low-quality embryos and those embryos cultured under suboptimal conditions (P<0.05). The TECS is proposed as a promising approach to improve embryo development and blastocyst formation by exposing embryos to mechanical stimuli similar to those in the Fallopian tube.

Blastocyst quality scoring based on morphologic grading correlates with cell number.

Blastocyst quality score (BQS), first reported by Rehman et al., is a numerical blastocyst-morphology grading system based on the criteria established by Gardner and Schoolcraft. We demonstrate a positive correlation between the calculated BQS score and cell number by staining thawed human embryos and suggest that BQS can be applied to evaluate culture systems clinically.

Increased plasma adrenomedullin in women with recurrent pregnancy loss.

OBJECTIVE: To evaluate vascular changes and uterine perfusion in women with recurrent pregnancy loss. METHODS: We measured plasma levels of adrenomedullin of 100 pregnant women in the midluteal phase of a nonpregnant cycle (control group: n = 62; recurrent pregnancy loss group: n = 38). We measured the pulsatility index (PI) in the uterine arteries by transvaginal pulsed Doppler ultrasonography at the same time. RESULTS: The plasma level of adrenomedullin in women with recurrent pregnancy loss (5.6 +/- 1.9, mean +/- standard deviation) was significantly higher (P >.001) than that in control women (3.6 +/- 1.7). Uterine arterial PI of women with recurrent pregnancy loss (2.70 +/- 0.47) was significantly higher (P >.001) than that in control women (2.09 +/- 0.39). Plasma level of adrenomedullin had a significant positive correlation with uterine arterial PI both in the control group (r =.58, P <.001) and in the recurrent pregnancy loss group (r =.78, P <.001). Both plasma adrenomedullin concentration (7.2 +/- 2.3) and uterine arterial PI (3.06 +/- 0.36) were significantly high in women with antiphospholipid antibodies. CONCLUSION: Plasma adrenomedullin may serve as a useful biochemical marker for recurrent pregnancy loss caused by impaired uterine perfusion.

Involvement of peroxynitrite in LPS-induced apoptosis of trophoblasts.

OBJECTIVE: To examine whether or not peroxynitrite was involved in trophoblastic apoptosis induced by a bacterial endotoxin, lipopolysaccharide (LPS). METHODS: Levels of nitrite/nitrate, stable metabolites of nitric oxide (NO), in culture medium of trophoblasts, were determined using Griess reagents. Trophoblastic apoptosis was identified morphologically and confirmed using in situ nick end labeling technique. The amount of nitrotyrosine, a footprint of peroxynitrite, was quantified by dot blotting. Statistical significance was determined by ANOVA. RESULTS: Treatment of trophoblasts with LPS leads to apoptosis accompanied by formation of NO and nitrotyrosine. Aminoguanidine, an inhibitor of NO synthase (NOS), reduced peroxynitrite formation and prevented apoptosis. Scavengers of peroxynitrite also prevented apoptosis in this culture model. CONCLUSION: Peroxynitrite was involved in trophoblastic apoptosis induced by LPS. Peroxynitrite scavengers or inhibitors of NOS may thus be candidate therapeutic agents for infectious diseases, which is associated with overproduction of NO and peroxynitrite.

Impaired uterine arterial blood flow in pregnant women with recurrent pregnancy loss.

OBJECTIVE: This study was undertaken to evaluate uterine perfusion, which regulates uterine receptivity, in women with recurrent pregnancy loss. METHODS: We evaluated the blood flow resistance in the uterine arteries of 104 pregnant women at 4 to 5 weeks' gestation by transvaginal pulsed Doppler ultrasonography (control group, n = 52; and recurrent pregnancy loss group, n = 52). Blood tests for antinuclear and antiphospholipid antibodies were also performed. RESULTS: The uterine arterial pulsatility index in the recurrent pregnancy loss group was significantly higher than that in the control group. Women with antinuclear or antiphospholipid antibodies had an elevated pulsatility index in the uterine artery, which is prominent in women with recurrent pregnancy loss. Coagulopathy and vascular dysfunction caused by autoantibodies may impair uterine perfusion. However, the uterine arterial pulsatility index in the recurrent pregnancy loss group was significantly higher than that in the control group even among women without antinuclear antibodies or among women without antiphospholipid antibodies. This observation strongly suggests that the uterine artery pulsatility index may be an independent index for recurrent pregnancy loss. CONCLUSIONS: The introduction of pulsed Doppler ultrasonography has provided the means for noninvasive evaluation of uterine impedance and may identify patients with recurrent pregnancy loss associated with impaired uterine perfusion.

Bisphenol A stimulates NO synthesis through a non-genomic estrogen receptor-mediated mechanism in mouse endothelial cells.

Biological actions of bisphenol A (BPA), an environmental chemical, have not been fully elucidated. We studied effect of BPA on nitric oxide (NO) synthesis in the murine endothelial cell line, MSS31. BPA (1-100 microM) increased nitrite/nitrate, a stable metabolites of NO, levels in culture medium of MSS31. However, Western blotting showed that the level of endothelial NO synthase protein was not increased by 16 h of treatment with BPA (10 microM). ICI 182,780 (10 microM), an estrogen receptor (ER) antagonist, suppressed BPA-induced NO synthesis while actinomycin D (1 microg/ml), a transcription inhibitor, or cycloheximide (40 microM), a protein synthesis inhibitor, exhibited no effect on BPA-induced NO synthesis. These results indicate that BPA stimulates NO synthesis through a non-genomic ER-mediated mechanism. Short-term effects of BPA on NO synthesis were weak but similar to 17beta-estradiol.

A long-term transdermal nitric oxide donor improves uteroplacental circulation in women with preeclampsia.

OBJECTIVE: To determine the effects of long-term transdermal administration (range, 4-30 days; mean +/- SD, 11.1+/-7.2 days) of isosorbide dinitrate, a nitric oxide donor, in preeclamptic women. METHODS: We studied uterine and fetoplacental circulation of 12 preeclamptic women with oligohydramnios and an elevated pulsatility index in the uterine arteries. RESULTS: Transdermal isosorbide dinitrate significantly suppressed the blood pressure of patients. Pulsed Doppler ultrasonography revealed that the average pulsatility index in the uterine arteries was significantly reduced by treatment with isosorbide dinitrate (P < .003). The average pulsatility index in the umbilical artery was also significantly reduced (P < .004). Furthermore, the size of the amniotic fluid pocket increased approximately 4-fold by treatment with isosorbide dinitrate. CONCLUSIONS: Long-term transdermal administration of isosorbide dinitrate improves fetoplacental circulation and may be effective therapy for avoiding maternal hypertension and oligohydramnios in some preeclamptic women.

The biological effects of antiadhesion agents on activated RAW264.7 macrophages.

The objective of this study is to determine the biological effects of various antiadhesion agents on macrophages, which play an essential role in wound healing and adhesion. To determine these effects, RAW264.7 macrophages were activated with lipopolysaccharide in the presence of antiadhesion agents: oxidized regenerated cellulose (oxyC), sodium hyaluronate (HA), dexamethasone (Dex), or chondroitin sulfate (CS). The release of nitric oxide (NO), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), or matrix metalloproteinases (MMPs) from RAW264.7 was measured. We found that oxyC reduced the release of NO, IL-6, MMP-2, and MMP-9, whereas it enhanced the release of VEGF. HA reduced the release of MMP-2, whereas it enhanced the release of VEGF and NO. HA exhibited no significant effect on the release of IL-6 or MMP-9. Dex reduced the release of NO, VEGF, IL-6, MMP-2, and MMP-9. CS reduced the release of VEGF, IL-6, and MMP-2, although it had no significant effect on the release of NO and MMP-9. Antiadhesion agents, which have been clinically used as physical barriers, modulated the functions of macrophages.