To compare the morphokinetic patterns of human embryos originating from vitrified oocytes (VITRI group) with those derived from freshly collected oocytes (CONTROL group) in oocyte donation cycles.
This is a retrospective observational study.
Embryolab Fertility Clinic, Embryology Lab, Thessaloniki, Greece.
The study included embryos from 421 vitrified oocytes from 58 oocyte donation cycles and 196 fresh oocytes from 23 oocyte donation cycles.
None.
Key time parameters, dynamic events, fertilization rates, degeneration rates, cleavage rates, blastocyst rates, pregnancy rates, clinical pregnancy rates, implantation rates, and live birth rates were estimated.
The mean survival rate of vitrified oocytes was 92.58% (±7.42%). Fertilization rates were significantly different between the 2 groups (VITRI group: 71.92% ± 20.29% and CONTROL group: 80.65% ± 15.22%) whereas the degeneration, cleavage, blastocyst, pregnancy, clinical pregnancy, ongoing pregnancy, implantation, and live birth rates were not significantly different between embryos derived from fresh or vitrified oocytes. Time-lapse analysis showed no significant difference in any key time parameter. However, when examining dynamic parameters, first cell cycle (CC1) (t2 − tPB2: from the second polar body extrusion (tPB2) up to 2 cells (t2)) showed a significant difference whereas CC1a (t2 − tPNf: from fading of the pronuclei (tPNf) up to 2 cells (t2)) was at the threshold of significance.
CC1 in vitrified oocytes exhibited a comparatively slower progression in contrast to fresh oocytes. Conversely, CC1a in vitrified oocytes demonstrated faster progression compared with fresh oocytes. It is worth noting that these temporary deviations had minimal impact on the subsequent development. Despite the clinical outcomes showing a decrease in the vitrified group, none of them reached statistical significance. This lack of significance could be attributed to the limited sample size of the study.
To identify the transcriptomic changes of ectopic lesions and eutopic endometrial tissues during the progression of endometriosis, we performed transcriptomic analysis in the eutopic endometrium and ectopic lesions.
Laboratory study.
Academic medical center.
Four fertile and 4 subfertile Pgrcre/+Rosa26mTmG/+ mice with endometriosis, and 4 sham mice for each group of endometriosis mice as control. These mice underwent either surgery to induce endometriosis or sham surgery. Fertile sham and mice with endometriosis were used 1 month after surgery, whereas subfertile ones were used 3 months after surgery.
Early and chronic effects of endometriosis on transcriptomics of ectopic lesions and eutopic endometrium.
RNA-sequencing analysis and identification of differentially expressed genes and pathways in the ectopic lesions and eutopic uteri from mice with endometriosis and sham mice at day 3.5 of pregnancy.
Our mouse model recapitulates the transcriptomic changes of ectopic lesions in humans. RNA-sequencing analysis was performed in ectopic lesions and eutopic uteri from mice with or without endometriosis during the progression of the disease. Estrogen activity, inflammation, angiogenesis, and fibrosis pathways were consistently elevated in all the ectopic lesions compared with eutopic endometrium. Cholesterol/glucose synthesis and stem cell pluripotency pathways were more enhanced in ectopic lesions from subfertile mice compared with their eutopic endometrium. Dysregulation of infiltration of macrophage, dendritic, T and B cells was validated with the use of immunohistochemistry in ectopic lesions. Multiple ligand–receptor pairs between the ectopic and eutopic endometrium were altered compared with the sham endometrium. Suppressed WNT and EGF pathways were only found in the eutopic endometrium from subfertile not fertile mice compared with sham.
Our mouse endometriosis model recapitulates the transcriptomics of ectopic lesions in humans. Our transcriptomic analysis during endometriosis progression in our mouse model will help us understand the pathophysiology of endometriosis.
To evaluate ovarian tissue and follicle integrity before and after slow freezing or vitrification and postthawing in vitro culture.
A laboratory study using bovine ovarian cortical tissue.
Academic laboratory.
Ovaries from healthy cattle.
Bovine ovarian cortical tissue was subjected to either slow freezing or vitrification and subsequent in vitro culture. Tissue and follicle integrity were assessed before and after cryopreservation and culture.
Hematoxylin and eosin staining was used to assess follicle stages, morphology, and stromal cell density. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to examine apoptosis, and Masson’s trichrome staining was used to evaluate collagen content in the stromal environment. Immunofluorescent labeling was used to localize and quantify connexin 37 (CX37) and Ki67 expression.
Regardless of previous cryopreservation, ovarian tissue culture resulted in a decreased percentage of primordial follicles and an increased percentage of primary follicles compared with fresh tissue, indicating that follicle activation was not negatively affected by cryopreservation. However, both culture and cryopreservation followed by culture decreased the percentage of normal preantral follicles compared with fresh tissue that had not been cultured. Culture and/or cryopreservation did not impact stromal cell number, but there was increased cell apoptosis in tissue that was cultured after vitrification compared with tissue that was not cultured. Tissue culture, regardless of cryopreservation, resulted in decreased collagen deposition. There were fewer follicles expressing CX37 in vitrified and thawed tissue compared with all other treatments. Cryopreservation and/or culture of ovarian tissue did not change the percentage of follicles that contained Ki67-positive granulosa cells or the percentage of Ki67-positive granulosa cells within those follicles.
Based on these data, we conclude that tissue cryopreservation followed by culture does not affect follicle activation and growth, but it decreases the proportion of viable follicles within the tissue. Slow freezing was superior to vitrification as indicated by a higher proportion of follicles with normal morphology, lower stromal cell apoptosis, and maintenance of CX37 expression postthawing and after culture.
To explore the taxonomic and predicted functional relationship between the urine microbiome and alterations of semen analysis (SA) parameters.
Cross-sectional study.
Academic medical center.
Men presenting for fertility evaluation or men presenting for vasectomy consultation with proven biological paternity were recruited and stratified on the basis of alterations, or lack thereof, in SA parameters.
Changes in the functional and taxonomic urine microbiome profiles of participants with or without alterations in SA parameters.
Seventy-three participants were included in our study. Men with abnormal sperm motility (N = 27) showed a nearly 50-fold higher abundance of Dialister micraerophilus compared with those with normal sperm motility (N = 46). This relationship persisted on canonical correlational analysis (r = 0.439). Men with abnormal sperm concentration (N = 20) showed a lower abundance of Enterococcus faecalis and Staphylococcus aureus, compared with those with normal sperm concentration (N = 53). The urine of participants with impaired sperm motility demonstrated dramatic differences in predictive functional profiles in pathways involved in oxidation–reduction balance and cell longevity.
Our findings underscore differences in the urinary microbiome and abnormalities in semen parameters, especially sperm motility. By incorporating predictive functional profiling, we also highlight possible mechanisms that may drive the observed differences in sperm parameters.
To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS).
Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA–sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes.
A laboratory study.
Three men with a diagnosis of obstructive azoospermia (age range, 30–40 years).
None.
Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue.
Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%–8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1−/doublesex and Mab-3 related transcription factor 1−/STRA8+ spermatogonia as well as SYCP3+/protamine 2− spermatocytes.
This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and in vitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.
To perform a comprehensive assessment of protamine (P) isoforms and modifications in human sperm with the aim of identifying how P modifications and isoforms are altered in men with reduced sperm motility and low sperm count.
Cross-sectional.
Academic medical center.
A total of 18 men with prior reported pregnancy and normozoospermia (normal sperm), 14 men from couples with infertility and asthenozoospermia (reduced sperm motility), and 24 men from couples with infertility and oligoasthenoteratozoospermia (low sperm count and motility and abnormal sperm morphology).
Not applicable.
Proteomic assessment using both top-down and bottom-up liquid chromatography mass spectrometry (MS) analysis.
A total of 13 posttranslational modifications were identified on P1 and P2 using bottom-up MS, including both phosphorylation and methylation. Top-down MS revealed an unmodified and phosphorylated isoform of P1 and the 3 major isoforms of P2, HP2, HP3, and HP4. Protamine 1 phosphorylation was overall higher in men with male factor infertility compared with those with normal semen analysis (40.5% vs. 32.6). There was no difference in P posttranslational modifications or isoforms of P2 in men with normal vs. abnormal fertility.
Human protamines bear a number of posttranslational modifications, with alterations in P1 phosphorylation noted in the setting of male factor infertility.
To compare salpingectomy and detorsion procedures and investigate the biochemical and histopathological changes in the fallopian tubes in the experimentally isolated fallopian tube torsion model in rats.
Experimental study.
Experimental surgery laboratory in a training and research hospital.
Twenty-seven Sprague-Dawley rats in the reproductive period.
Group 1, control group (n = 6); group 2, bilateral total salpingectomy group after 4 hours of tubal ischemia (n = 7); group 3: 4 hours of bilateral tubal ischemia plus 1 week of reperfusion (n = 7); and group 4, 4-hour period of bilateral tubal ischemia plus 30 days of reperfusion (n = 7). A 22-gauge catheter was administered before and after surgery using methylene blue through the uterine horn of the rat to evaluate tubal patency.
Preoperative and postoperative serum antimüllerian hormone (AMH) levels, histopathological examination of the rat tuba uterine and histopathological damage scores, antioxidant compounds (superoxide dismutase [SOD], catalase, and glutathione peroxidase [GSH-Px]), and oxidative stress end product levels (malondialdehyde [MDA] and 8-hydroxy-2′-deoxyguanosine [8-OHdG]).
Although a significant difference was observed in the tissue SOD, GSH-Px, MDA, and 8-OHdG values, no significant difference was observed between the groups in serum samples. The tissue SOD and tissue GSH-Px levels in group 2 significantly decreased, and a significant increase was observed in the tissue MDA and 8-OHdG values in group 2. Among the histopathological parameters, epithelial changes, vascular congestion, and the total fallopian tube mean damage score of 4 showed a significant decrease in group 4. When the methylene blue transitions before and after ischemia-reperfusion injury were compared, the values of the methylene blue transition after ischemia-reperfusion injury in groups 2–4 significantly decreased. When the serum AMH levels were analyzed, the postoperative AMH value in group 2 significantly increased.
This study reveals that biochemical and histopathological improvement is observed in the fallopian tube tissues gradually when the detorsion procedure is performed for the necrotized tubal tissue instead of salpingectomy. Although there is restoration of epithelial integrity after reperfusion, tubal passage remains absent.
This study was approved by the Local Ethics Committee for Animal Experiments of the Health Sciences University, Istanbul Hamidiye Medicine Faculty (approval number 27.05.2022-9269). The study followed the ethics standards recommended by the Declaration of Helsinki.
To investigate the long-term effects of in utero taxane exposure on exposed daughters’ ovarian reserve and reproductive potential.
Pregnant dams were treated with a single, human-relevant animal-equivalent dose of saline, docetaxel, or paclitaxel at embryonic day 16.5. In utero-exposed daughters were aged to multiple postnatal time points for ovarian and endocrine analysis or were bred to assess fertility and fecundity. Granddaughters of treated dams were assessed also for ovarian follicle composition and atresia.
Laboratory study.
C57BL/6 mice.
In utero exposure to saline, docetaxel, or paclitaxel.
Ovarian follicle composition, rates of follicle atresia, and rates of multioocyte follicles were analyzed in all exposure groups. Serum hormone levels and oocyte retrieval outcomes following ovarian hyperstimulation were also assessed. Finally, animals from all exposure groups were bred with the number of litters, pups per litter, live births, interlitter time interval, and age at the last litter analyzed.
We found that docetaxel and paclitaxel exposure in utero results in ovarian toxicity later in life, significantly affecting folliculogenesis as well as increasing the rate of follicular abnormalities, including follicle atresia and multioocyte follicles. Furthermore, viability staining indicates that the ovaries of daughters exposed to taxanes in utero demonstrate a significantly higher number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive follicles. Hormone measurements also revealed that serum follicle-stimulating hormone concentration was significantly altered in taxane-exposed daughters, with the ratio of luteinizing hormone to follicle-stimulating hormone significantly elevated, specifically after paclitaxel exposure, coincident with the inability of these animals to properly respond to ovarian stimulation. Breeding studies over the course of a year also suggest that these taxane-exposed mice are fertile, although the duration of their fertility is shortened and they produce significantly fewer litters. Finally, ovarian effects are apparent in granddaughters of mice treated with docetaxel, suggesting persistent and multigenerational effects of taxane exposure.
Our studies demonstrate that in utero exposure to taxane-based therapy during late gestation has a significant effect on the long-term reproductive health of exposed daughters (as well as their daughters) and will be instrumental in helping clinicians better understand which chemotherapies for maternal malignancy are least detrimental to a developing fetus.
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