Purpose
This study aimed to evaluate the value of long-read sequencing for preimplantation haplotype linkage analysis.
Methods
The genetic material of the three β-thalassemia mutation carrier couples ...was sequenced using single-molecule real-time sequencing in the 7.7-kb region of the HBB gene and a 7.4-kb region that partially overlapped with it to detect the presence of 17 common HBB gene mutations in the Chinese population and the haplotypes formed by the continuous array of single-nucleotide polymorphisms linked to these mutations. By using the same method to analyze multiple displacement amplification products of embryos from three families and comparing the results with those of the parents, it could be revealed whether the embryos carry disease-causing mutations without the need for a proband.
Results
The HBB gene mutations of the three couples were accurately detected, and the haplotype linked to the pathogenic site was successfully obtained without the need for a proband. A total of 68.75% (22/32) of embryos from the three families successfully underwent haplotype linkage analysis, and the results were consistent with the results of NGS-based mutation site detection.
Conclusion
This study supports long-read sequencing as a potential tool for preimplantation haplotype linkage analysis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Although pre-treatment with a GnRH agonist can reduce the size of adenomyosis lesions, the supra-physiological hormone level induced by controlled ovarian hyperstimulation (COH) may negate the ...usefulness of the GnRH agonist in patients with adenomyosis lesions, leading to continued poor outcomes in fresh embryo transfer cycles during
fertilization (IVF). It is unclear whether GnRH agonist pre-treatment before starting the long GnRH agonist protocol for IVF/ICSI (intracytoplasmic sperm injection) can improve cumulative live birth rate (CLBR) of infertile women with adenomyosis.
In this retrospective cohort study, a total of 374 patients diagnosed as adenomyosis (477 cycles) underwent IVF/ICSI with long GnRH agonist protocol with or without GnRH agonist pre-treatment between January 2009 and June 2018. Logistic regression was used to assess the association between GnRH agonist pre-treatment and pregnancy outcome after adjusting for confounding factors.
The live birth rate in fresh embryo transfer cycles was higher in the non-pre-treatment group than in the GnRH agonist pre-treatment group (37.7 vs. 21.2%,
= 0.028); the adjusted odds ratio (OR) for the long agonist protocol without pre-treatment was 1.966 (95% CI: 0.9-4.296,
= 0.09). The CLBR was higher in the non-pre-treatment group than in the GnRH agonist pre-treatment group (40.50 vs. 27.90%,
= 0.019); the adjusted OR for the long agonist protocol without pre-treatment was 1.361 (95% CI: 0.802-2.309,
= 0.254).
Our results indicated that GnRH agonist pre-treatment before starting the long GnRH agonist protocol does not improve the live birth rate in fresh embryo transfer cycles or CLBR in infertile women with adenomyosis after IVF/ICSI treatment when compared to that in non-pre-treated patients. A subsequent prospective randomized controlled study is needed to confirm these results.
Abstract
Recombination is essential for physical attachments and genetic diversity. The Han Chinese population is the largest ethnic group worldwide, therefore, the construction of a genetic map ...regarding recombination for the population is essential. In this study, 164 and 240 couples who underwent preimplantation genetic testing for monogenic diseases or segmental rearrangement were included in the analysis. Blastocysts and probands from couples who underwent preimplantation genetic testing for monogenic diseases by single nucleotide polymorphism array were included for recombination analysis. The location of recombination was determined from haplotype phase transitions in parent-offspring pairs at loci where the parents were heterozygous. The genetic map for Chinese in vitro fertilization embryos was constructed by the expectation–maximization algorithm with chip-level data. Our results confirmed that homologous recombination occurred more often in maternal chromosomes, and the age effect was more significant in maternal homologous recombination. A total of 6,494 homologous recombination hotspots (32.3%) were identified in genes of Online Mendelian Inheritance in Man. A uniform association between homologous recombination and aneuploidy was not established. In addition, carriers with identified breakpoints of reciprocal translocations were analyzed, and locations of breakpoints were found partly overlapped with homologous recombination hotspots, implying a possible similar mechanism behind both events. This study highlights the significance of constructing a recombination map, which may improve the accuracy of haplotype analysis for preimplantation genetic testing for monogenic diseases. Overlapping locations of translocation and recombination are worthy of further investigation.
To compare the two GnRH-a protocols (long GnRH-a protocol and short GnRH-a protocol) for ovarian stimulation in IVF/ICSI cycles in patients of various age ranges.
A total of 5662 IVF-ET/ICSI cycles ...from 2010 to 2013 were retrospectively identified. The cycles were divided into two groups: a long protocol group and short protocol group. In each group, the patients were divided into four age ranges: <31 years, 31 to 35 years, 36 to 40 years, and >40 years. The duration of stimulation, total dose of Gn, implantation rate and pregnancy rate were compared.
The total dose of Gn was significantly higher, and the duration of stimulation was significantly longer, in the long protocol group than in the short protocol group for all age ranges (P<0.05). If the patients were of the same age range, the number of oocytes retrieved, MII oocytes, and high-quality embryos in the long protocol group were all significantly greater than those in the short protocol group (P<0.05). In the long protocol group, the clinical pregnancy rates of the four age ranges were 52.76%, 44.33%, 36.15% and 13.33%, respectively, which were significantly higher than those in the short protocol group (33.33%, 24.58%, 22.49% and 8.72%, respectively; P<0.05). The same trend was also found in the implantation rates of the four age ranges. As the age increased, the clinical pregnancy and implantation rates, as well as the number of oocytes retrieved, MII oocytes, and high-quality embryos, of the long protocol group significantly decreased (P<0.05).
Our study demonstrated that regardless of patient age, the long protocol was superior to the short protocol in terms of the number of retrieved oocytes, as well as the implantation and pregnancy rates.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aim
To determine whether single endometrial polyp (EP) or multiple EP (polyp number ≥ 6) are associated with chronic endometritis (CE).
Methods
From June 2017 to December 2018, this study enrolled a ...total of 277 patients, including 92 patients with multiple EP, 82 patients with a single EP and 103 patients without polyps who underwent hysteroscopic examination and polypectomy. Polyps and endometrium samples were obtained and subjected to immunohistochemistry for CD138 to identify plasma cells and CE was diagnosed as CD138‐positive plasma cells greater than or equal to 5/high power field. The prevalence of CE was compared and analyzed using the logistic regression model.
Results
All baseline parameters were comparable among the three groups except that the prevalence of abnormal uterine bleeding (AUB) was much higher in both polyp groups than the non‐polyp control. The prevalence of CE was significantly higher in the multiple EP group than in the single EP group (58.7% vs 28.0%, P < 0.001). There was no difference on the prevalence of CE between the single EP and the non‐polyp groups (28.0% vs 29.1%, P = 0.872). Multivariable analysis revealed that AUB (adjusted OR 2.81, 95% CI 1.35–5.87) and multiple EP (adjusted OR 2.58, 95% CI 1.38–4.82) were independently associated with CE, while the single EP did not increase the odds of CE compared to the non‐polyp group (adjusted OR 0.74, 95% CI 0.38–1.45).
Conclusion
Multiple EP were positively associated with CE among reproductive‐aged women, suggesting a possible hidden etiopathogenetic link between chronic inflammation and multiple EP.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
In preimplantation genetic testing for aneuploidy (PGT-A), appropriate evaluation of mosaic embryos is important because of the adverse implications of transferring embryos with high-level mosaicism ...or discarding those with low-level mosaicism. Despite the availability of multiple reliable techniques for PGT-A, data comparing the detection of mosaicism using these techniques are scarce. To address this gap in the literature, we compared the detection ability of the two most commonly used PGT-A platforms, next-generation sequencing (NGS) and the single-nucleotide polymorphism (SNP) array, for mosaic embryos.
We retrospectively reviewed the data of PGT-A or preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) conducted at our center from January 2018 to October 2020, and selected blastocysts that underwent aneuploidy screening with both an SNP array and NGS. Trophectoderm biopsy, multiple displacement amplification (MDA), and aneuploidy screening with an SNP array were conducted on the enrolled blastocysts. When the SNP array indicated mosaicism, NGS was performed on the corresponding MDA product for verification. Among the 105 blastocysts diagnosed with mosaicism with the SNP array, 80 (76.19%) showed mosaicism in NGS, with complete and partial concordance rates of 47.62% (50/105) and 18.10% (19/105), respectively. The complete discordance rate of the two platforms was 34.29% (36/105). That is, 10.48% (11/105) of the blastocysts were diagnosed with completely different types of mosaicism with the two platforms, while 13.33% (14/105) and 10.48% (11/105) of the embryos diagnosed as showing mosaicism with SNP were detected as showing aneuploidy and euploidy with NGS, respectively.
The consistency of NGS and the SNP array in the diagnosis of embryo mosaicism is extremely low, indicating the need for larger and well-designed studies to determine which platform is more accurate in detecting mosaic embryos.
Full text
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A key step in embryo implantation is the adhesion to and invasion of the endometrium by the blastocyst trophectoderm. The envelope proteins of HERV-W and -FRD (human endogenous retrovirus-W and ...-FRD), syncytin-1 and syncytin-2, are mainly distributed in the placenta, and play important roles in the development of the placenta. The placenta originates from the trophectoderm of the blastocyst. It is unclear whether the envelope proteins of HERV-W and -FRD have an effect on the development of the trophectoderm and whether they have any association with the implantation of the blastocyst.
The whole-genome amplification products of the human blastocyst trophectoderm were used to measure the copy number of syncytin-1 and syncytin-2 using real time qPCR. In addition, clinical data associated with the outcome of pregnancies was collected, and included age, body mass index (BMI), basic follicle stimulating hormone(bFSH), rate of primary infertility and oligo-astheno-teratospermia, the thickness of the endometrium on the day of endometrial transformation, the levels of estrogen and progestin on the transfer day, the days and the morphological scores of the blastocysts. The expression of mRNA and the copy numbers of syncytin-1 and syncytin-2 in H1 stem cells, and in differentiated H1 cells, induced by BMP4, were measured using real time qPCR.
The relative copy number of syncytin-1 in the pregnant group (median: 424%, quartile: 232%-463%,
< 0.05) was significantly higher than in the non-pregnant group (median: 100%, quartile: 81%-163%). There was a correlation (
= 0.681,
< 0.001) between the copy number of syncytin-1 and blastocyst implantation after embryo transfer. As the stem cells differentiated, the expression of NANOG mRNA decreased, and the expression of caudal type homeobox 2(CDX2) and
-human chorionic gonadotropin (
-hCG) mRNAs increased. Compared to the undifferentiated cells, the relative expression of the syncytin-1 mRNA was 1.63 (quartile: 0.59-6.37,
> 0.05), 3.36 (quartile: 0.85-14.80,
> 0.05), 10.85 (quartile: 3.39-24.46,
< 0.05) and 67.81 (quartile: 54.07-85.48,
< 0.05) on day 1, 3, 5 and 7, respectively, after the differentiation. The relative expression of syncytin-2 was 5.34 (quartile: 4.50-10.30), 7.90 (quartile: 2.46-14.01), 57.44 (quartile: 38.35-103.87) and 344.76 (quartile: 267.72-440.10) on day 1, 3, 5 and 7, respectively, after the differentiation (
< 0.05). The copy number of syncytin-1 increased significantly during differentiation.
Preceding the transfer of frozen embryos, the increased copy number of syncytin-1 in the blastocyst trophectoderm was associated with good outcomes of pregnancies.
Aim
This retrospective study aimed to evaluate the predictive value of serum cancer antigen 125 (CA125) levels before hormone replacement therapy on pregnancy outcomes in women with adenomyosis ...undergoing frozen embryo transfer.
Methods
A total of 509 women with adenomyosis were screened and 84 patients receiving a total of 114 cycles of frozen embryo transfer were included, based on the inclusion and exclusion criteria. Patients were divided into two groups based on their CA125 levels (≤ or >35 IU/mL) before hormone replacement therapy. The basic characteristics and main outcomes of the two groups were compared. Receiver operating characteristic curve and subgroup analyses were also conducted.
Results
There were no significant differences in clinical outcomes of frozen embryo transfer cycles in patients with different serum CA125 levels before hormone replacement therapy. Receiver operating characteristic curve analysis demonstrated that CA125 levels before hormone replacement therapy were not predictive of clinical pregnancy outcomes.
Conclusions
Serum CA125 levels before hormone replacement therapy are not associated with the clinical outcomes of frozen embryo transfer among women with adenomyosis.
This study aimed to evaluate the feasibility and necessity of using fluorescence Gap-polymerase chain reaction combined with haplotype analysis in preimplantation genetic testing for SEA-type ...α-thalassemia.
A total of 26 preimplantation genetic testing biopsy cycles were performed in 25 families from June 2021 to February 2022. All couples were carriers of SEA-type α-thalassemia. Fluorescent Gap-polymerase chain reaction was used for detecting fragment deletion. Subsequently, according to the results of polymerase chain reaction, reference embryos were identified to establish haplotype using single nucleotide polymorphism array, and aneuploidy was screened simultaneously. In cases wherein the polymerase chain reaction results were inconsistent with the haplotype results, the reasons were investigated, either by retest of the biopsied samples or rebiopsy of the embryo.
Among the 172 embryos, 162 had consistent results when tested using both methods, resulting in a consistency rate of 94.2%. Conversely, 10 embryos had inconsistent results, mainly due to chromosome 16 aneuploidy (
= 7), allele dropout in Gap-polymerase chain reaction (
= 2), or incorrect haplotype due to poor sample amplification quality (
= 1). The clinical pregnancy rate of each frozen-thawed embryo transfer was 57.7% (15/26). Six families underwent prenatal diagnosis, which confirmed the results of preimplantation genetic testing.
Fluorescent Gap-polymerase chain reaction combined with haplotype analysis is feasible and necessary for SEA-type α-thalassemia preimplantation genetic testing.
Background Ovarian hyperstimulation syndrome is an iatrogenic complication of controlled ovarian stimulation. Early ovarian hyperstimulation syndrome occurs during luteal phase of controlled ovarian ...stimulation within 9 days after human chorionic gonadotropin trigger and reflects an acute consequence of this hormone on the ovaries. Late ovarian hyperstimulation syndrome occurs 10 or more days after human chorionic gonadotropin trigger and reflects increased endogenous human chorionic gonadotropin levels following pregnancy. Human chorionic gonadotropin stimulates granulosa-lutein cells to produce vascular endothelial growth factor messenger RNAs, which in turn raises serum vascular endothelial growth factor concentration and increases vascular permeability in women with ovarian hyperstimulation syndrome. Efforts to reduce the incidence and severity of ovarian hyperstimulation syndrome after oocyte retrieval, and in particular primary prevention efforts, are vital to prevent thrombogenesis and other serious complications. Objective The objective of the study was to compare the efficacy of letrozole, an aromatase inhibitor, with aspirin in primary prevention of early ovarian hyperstimulation syndrome and to compare vascular endothelial growth factor levels between groups. Study Design Participants in this prospective randomized trial included 238 participants undergoing cryopreservation of the whole embryos after oocyte retrieval with at least 1 of the following high-risk factors for ovarian hyperstimulation syndrome: oocyte retrieval ≥25; estradiol level ≥5000 pg/mL on the day of human chorionic gonadotropin administration; and clinical or ultrasonographic evidence of ovarian hyperstimulation syndrome on the day of oocyte retrieval, such as ultrasonographic evidence of ascites. After human chorionic gonadotropin triggering, experimental (119 cases) and control (119 cases) groups received letrozole and aspirin, respectively, for 5 days. The 5 categories of ovarian hyperstimulation syndrome include no, yes-mild, yes-moderate, yes-severe, and yes-critical. The primary outcome was the incidence and severity of early ovarian hyperstimulation syndrome. The secondary outcome included vascular endothelial growth factor level both on the second and seventh day after the human chorionic gonadotropin trigger, and clinical and laboratory features of ovarian hyperstimulation syndrome symptoms. Results The incidence of ovarian hyperstimulation syndrome was significantly higher in women receiving aspirin, compared with letrozole (90.2% vs 80.4%, P = .044). Moderate and severe ovarian hyperstimulation syndrome was also higher in the aspirin group, 45.1%, compared with the letrozole group, 25.0% ( P = .002). Moreover, the duration of luteal phase was shortened in letrozole group compared with aspirin group (8.1 ± 1.1 days vs 10.5 ± 1.9 days, P < .001). The vascular endothelial growth factor level was significantly higher in the letrozole-treated group than aspirin-treated group (0.49 ± 0.26 vs 0.42 ± 0.22, P = .029). Conclusion Letrozole was more effective than aspirin in decreasing the incidence of moderate and severe early-onset ovarian hyperstimulation syndrome. Our results indicate that ovarian hyperstimulation syndrome might be caused through a luteolytic effect rather than through modulation of vascular endothelial growth factor, racing by a decline in estradiol and termination of early-onset ovarian hyperstimulation syndrome in advance in high-risk women with cryopreservation of the whole embryos.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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