Female Embryos Research Articles

Nanopore-Based Sequencing of the Full-Length Transcriptome of Male and Female Cleavage-Stage Embryos of the Chinese Mitten Crab (Eriocheir sinensis)

The cleavage stage plays a crucial role in embryo development, characterized by a swift surge in cell proliferation alongside the accurate genetic material transmission to offspring. To delve into the characteristics of sex development during the cleavage stage of embryos, we generated the full-length transcriptome of Eriocheir sinensis male and female cleavage-stage embryos using Oxford Nanopore Technologies (ONT). Notably, this investigation represents the first sequencing effort distinguishing between genders in E. sinensis embryos. In the transcriptome structure analysis, male and female cleavage-stage embryos, while not clustered, exhibited a comparable frequency of alternative splicing (AS) occurrences. We also successfully identified 2875 transcription factors (TFs). The quantitative analysis showed the top 150 genes, in which the highly expressed genes in male embryos predominantly related to protein synthesis and metabolism. Further investigation unveiled 500 differentially expressed genes (DEGs), of which 7 male-biased ribosomal protein genes (RPGs) were particularly noteworthy and further confirmed. These analyses suggest that there may be a more active protein synthesis process in male E. sinensis cleavage-stage embryos. Furthermore, among the 2875 identified TFs, we predicted that 18 TFs could regulate the differentially expressed RPGs, with most TFs belonging to the zf-C2H2 and Homeobox families, which are crucial for embryonic development. During the cleavage stage of E. sinensis, the differential RPGs between genders were intricately linked to energy metabolism. We proposed that these RPGs exert regulatory effects on gene expression in E. sinensis, thereby regulating the difference of development between male and females. Our research sheds light on the developmental mechanisms of E. sinensis during the embryo stage and establishes a groundwork for a deeper understanding of sex development in E. sinensis. The results also provide comprehensive full-length transcriptome data for future gene expression and genetic studies in E. sinensis.

High-altitude hypoxia exposure alters follicular metabolome and oocyte developmental potential in women.

To explore the effects of high-altitude hypoxia on the microenvironment of oocyte development and fertilization potential, we compared the metabolomic patterns of follicular fluid from women living in different altitude areas and traced their oocyte maturation and subsequent development. A total of 315 clinical cases were collected and divided into three groups according to their residence altitudes: 138 cases in low-altitude (< 2300m) group, 100 cases in middle-altitude (2300-2800m) group and 77 cases in high-altitude (> 2800m) group. The clinical outcomes were statistically estimated, including hormonal level, oocyte maturation, in vitro fertilization, and embryo development. Meanwhile, a metabolomic analysis was performed on the follicular fluid of women from different groups using ultra-high-performance liquid chromatography and high-resolution mass spectrometry and differential metabolites were analyzed through the KEGG pathway. The clinical data indicated that the physical condition and reproductive hormone secretion were similar among different groups. Although personalized gonadotropin-releasing hormone strategies were applied, the numbers of antral follicles and obtained oocytes were not impacted by the residence altitude change. In in vitro culture, the maturing rate, fertility rate and cleavage rate of high-altitude group were compared with the other groups. However, the rates of high-quality embryo, formative blastocyst, and available blastocyst were gradually decreased with the rise of residence altitude. Metabolome analysis identified 1193 metabolites in female follicular fluid. Differential analysis indicated that metabolic components in follicular fluid were remarkably changed with the elevation of residence altitude. These differential metabolites were closely related with amino acid metabolism, protein digestion and absorption, oocyte meiosis and steroid biosynthesis. The residence altitude alters the microenvironment of follicular fluid, which could damage the oocyte developmental potential. This study provides diagnostic basis and therapeutic targets for research on female oocyte and embryo development.

The development of the external genitals in female human embryos and foetuses. Part 2: Vaginal vestibule, anal canal, perineal raphe and perineal cutaneous muscles.

Concomitant with the rupture of the cloacal membrane, the perineal skin epithelium thickens (see accompanying article). In this study, we establish in female embryos and foetuses that the thick skin area divides into ventral and dorsal areas at ~14 weeks and gradually becomes restricted to the vaginal vestibule and anal canal thereafter. The dense mesenchymal core of the labia minora, which forms at ~8 weeks, extends dorsally to the anal canal as a midline reinforcement. The skin epithelium overlying this reinforcement is much thinner than the flanking 'thick skin', and is supported by an interrupted basement membrane, which implies epithelial-mesenchymal transformation of the thin midline epithelium and the subsequent establishment of the perineal raphe by the merging of the adjacent thick epithelium. Meanwhile, the anogenital distance in the perineum increases rapidly in length. Perhaps as a consequence, the labia minora cover only the ventral third of the vaginal vestibule at 20 weeks. The endodermal ducts of Bartholin's glands are identifiable at 7 weeks, while acini form at ~12 weeks. The vestibular bulbs become identifiable at ~10 weeks and form vascular networks after ~14.5 weeks. After the rupture of the cloacal membrane, the diameter of the junction of the dorsal cloaca with the anal canal is just a pinhole but widens dorsoventrally after the 7th week. The cutaneous muscles of the perineal area form as a ventrally open U-shaped mesenchymal mass, from which the anal sphincter and bulbospongiosus muscle develop. In conclusion, our findings show that thick skin epithelium persists in the vaginal vestibule and anal canal.

The development of the external genitals in female human embryos and foetuses. Part 1: Perineal thick skin, clitoris and labia.

Concomitant with the rupture of the cloacal membrane in the 6th week of development, the intermediate layer of the perineal-skin epithelium thickens. We investigated its distribution and the development of the corresponding subcutaneous compartments in serial sections of female human embryos and foetuses and prepared 3D reconstructions to establish topographic relations. The thick-skin area becomes restricted to the outlets of the genital and intestinal tracts. The clitoris and labia majora become identifiable at ~7 weeks. The mesenchymal mass inside the clitoris soon divides into the glans and the cavernous bodies. The clitoral hood forms between 10 and 14 weeks as a fold of tissue that extends from proximal to distal over the glans. Due to the caudal bending of the clitoral shaft, the labia majora gradually cover the clitoris after ~14 weeks. The labia minora form at ~8 weeks from the ridges of thick-skin epithelium that flank the genital exit. They are continuous ventrolaterally with the clitoral hood and ventromedially with the apex of the cavernous body. Dorsally, their dense subcutaneous mesenchymal core extends to the anal canal. Between 8 and 14 weeks, the urethra lengthens axially, while the vaginal vestibule extends ventrally. In this period, the urethral plate of female embryos is mitotically active but does not increase in volume, which suggests that it contributes to vestibular growth. In conclusion, we observed a temporal correlation between the development of the thick-skin epithelium and that of the external genitals, with a distribution that is reminiscent of the dihydrotestosterone-sensitive skin.

A Novel Trichinella spiralis Galectin Strengthens the Macrophage ADCC Killing of Larvae via Driving M1 Polarization.

Galectin recognizes β-galactosides through its carbohydrate recognition domains (CRDs). This study aimed to determine the biological features of a novel Trichinella spiralis galectin (galactoside-binding lectin family protein, TsGLFP) and its role in driving macrophage M1 polarization and enhancing ADCC killing of larvae. TsGLFP belongs to the galectin family and has two CRDs. The complete TsGLFP cDNA sequence was cloned and then expressed in Escherichia coli BL21. The results of qPCR, Western blot, and indirect immunofluorescence tests (IIFTs) revealed that TsGLFP was expressed in various stages of T. spiralis worms and principally localized at the cuticle and around the female embryos of the nematode. rTsGLFP had the function of agglutinating mouse erythrocytes, and this agglutination activity could be inhibited by lactose. After the mouse macrophage RAW264.7 was incubated with rTsGLFP, the expression level of the M1 genes (iNOS, IL-6, and TNF-α) and NO production were obviously increased. After incubating macrophages with rTsGLFP, there was a noticeable rise in the expression levels of p-IκB-α and p-NF-κB p65. Additionally, rTsGLFP enhanced the macrophage's ability to kill newborn larvae by ADCC cytotoxicity. When the macrophages were pretreated with the specific p-NF-κB p65 inhibitor PDTC, and then stimulated with rTsGLFP, the expression levels of iNOS, NO, and p-NF-κB p65 and the macrophages' ADCC cytotoxicity were distinctly decreased. These findings indicated that rTsGLFP enhanced the macrophage ADCC killing of larvae by driving M1 polarization through activating the NF-κB pathway.

Female embryos are more likely to die than males in a wild mammal

Female embryos are more likely to die than males in a wild mammal

Impact of overweight and obesity on live birth rate in single euploid frozen embryo transfers: the key is in the preparation protocol!

Research questionDoes endometrial preparation using a natural cycle (NC) lead to higher live birth rates (LBR) in single euploid frozen embryo transfers (seFET) compared to programmed cycles (PC), for women across normal, overweight, and obese body mass index (BMI) categories? DesignRetrospective study of 845 seFETs. Patients were stratified by BMI into 3 categories (Normal weight, Overweight, and Obesity Class I/II). Outcome was LBR. ResultsA total of 845 seFET cycles of 688 couples were included in the analysis. After achieving covariate (female age, AMH, embryo quality and infertility type) balance in each stratum, the effective sample size was 481 and 262 for the PC and NC, respectively. The PC approach, in which patients received vaginal luteal phase support with 3×100 mg micronized vaginal progesterone (MVP)/day, were associated with significantly lower LBR in the weighted regression analysis of the cohort (RR: 0.80, 95%CI: 0.73 – 0.88, p<0.001), compared to the NC approach. The effect was significantly modified by BMI (P=0.003). While the effect was significant for all BMI categories, the reduction in live birth was less pronounced in patients with normal or overweight BMI (RR: 0.87, 95%CI: 0.78-0.97, p=0.014) compared to patients with class I/II obesity (RR: 0.61, 95%CI: 0.49-0.75, p<0.001). ConclusionA natural cycle endometrial preparation approach leads to overall better live birth rates in single euploid FET, and the most significant difference is observed in women with higher BMI. Furthermore, overweight / obese patients in a PC may require a higher dosage of progesterone for luteal phase support.

A genetically small fetus impairs placental adaptations near term.

The placenta is a gatekeeper between the mother and fetus, adapting its structure and functions to support optimal fetal growth. Studies exploring adaptations of placentae that support the development of genetically small fetuses are lacking. Here, using a mouse model of impaired fetal growth, achieved by deleting insulin-like growth factor 2 (Igf2) in the epiblast, we assessed placental nutrient transfer and umbilical artery (UA) blood flow during late gestation. At embryonic day (E) 15.5, we observed a decline in the trans-placental flux of glucose and system A amino acids (by using 3H-MeG and 14C-MeAIB), proportionate to the diminished fetal size, whereas UA blood flow was normal. However, at E18.5, the trans-placental flux of both tracers was disproportionately decreased and accompanied by blunted UA blood flow. Feto-placental growth and nutrient transfer were more impaired in female conceptuses. Thus, reducing the fetal genetic demand for growth impairs the adaptations in placental blood flow and nutrient transport that normally support the fast fetal growth during late gestation. These findings have important implications for our understanding of the pathophysiology of pregnancies afflicted by fetal growth restriction.

Development of a PCR-based method to identify fetal sex during IVF cycles.

One of the most recognizable cases of preimplantation genetic diagnosis (PGD) is X-linked diseases. Diagnosis of fetal sex is essential for couples who are known to be at risk of some X-linked disorders. The objective of this study was to discriminate between female (XX) and male (XY) embryos by detecting sex chromosomes-specific sequences in spent culture medium and comparing these results to PGD/CGH array results. It may open new window for the development of a non-invasive PGD method. 120 Embryo's spent media from Day 3 and Day 5 embryos were collected. Modified phenol-chloroform solution was used for DNA extraction from spent media. Sex determination was performed using SRY, TSPY and AMELOGENIN evaluation through quantitative polymerase chain reaction (q-PCR) method. IBM SPSS and MedCalc were used for statistical analyses to compare sex determination of embryos by spent medium with PGD/CGH array results. Culture time was demonstrated to increase the DNA amount among day 5 embryos culture medium samples. Non-invasive PGD by means of spent culture medium gave a sensitivity, specificity, positive predictive value and negative predictive value of 100% for sex determination. Results of sex determination using spent medium by q-PCR were consistent with the results of PGD/CGH array. Improvements in cell-free DNA extraction and PCR amplification procedures provide us an effective method to perform a PGD test without biopsy in the future, especially about X-linked diseases.

Transvaginal three-dimensional ultrasound assessment of embryonic genital tubercle at 8-10+6weeks of gestation.

To assess embryonic genital tubercle using transvaginal three-dimensional (3D) ultrasound at 8-10+6weeks of gestation. One-hundred and two transvaginal 3D ultrasound scans were performed for first-trimester dating at 8-10+6weeks of gestation. The genital tubercle angle (GTA) and genital tubercle length (GTL) were measured with a mid-sagittal view of the embryo using the 3D ultrasound multiplanar mode. Intra- and inter-observer agreements regarding GTA and GTL were also assessed with Bland-Altman plots and intra- and inter-correlation coefficients. There were no significant differences in GTA between male and female embryos at 8, 9, 10weeks, or 8-10+6weeks of gestation, respectively. There were also no significant differences in GTL between male and female embryos at 8, 9, 10weeks, or 8-10+6weeks of gestation, respectively. However, GTL increased linearly with advancing gestation (r=0.8276, p<0.00001). Mean GTL (SD) values at 8, 9, and 10weeks were 0.833 mm (0.274), 1.623 mm (0.262), and 2.152 mm (0.420), respectively (p<0.001). Intra- and inter-reproducibilities of GTA and GTL were excellent. The intra- and inter-correlation coefficients of GTA and GTL were 0.964 and 0.995, and 0.996 and 0.9933, respectively. The genital tubercle could be identified using transvaginal 3D ultrasound at 8-10+6weeks of gestation. However, sex differentiation could not be performed at this age. The genital tubercle linearly developed with advancing gestation during the mid-first trimester of pregnancy.

Variant analysis and PGT-M of OTC gene in a Chinese family with ornithine carbamoyltransferase deficiency

BackgroundOrnithine carbamoyltransferase deficiency (OTCD) is a kind of X-linked metabolic disease caused by a deficiency in ornithine transcarbamylase leading to urea cycle disorders. The main reason is that the OTC gene variants lead to the loss or decrease of OTC enzyme function, which hinders the ammonia conversion to urea, resulting in hyperammonemia and severe neurological dysfunction. Here, we studied one Chinese family of three generations who consecutively gave birth to two babies with OTCD. This study aims to explore the pathogenicity of two missense variants in the OTC gene and investigate the application of preimplantation genetic testing for monogenic (PGT-M) for a family troubled by Ornithine carbamoyltransferase deficiency (OTCD).MethodsThe retrospective method was used to classify the pathogenicity of two missense variants in the OTC gene in a family tortured by OTCD. Sanger sequencing was used to validate the variants in the OTC gene, and then the pathogenicity of variants was confirmed through family analysis and bioinformatics software. We used PGT-M to target the OTC gene and select a suitable embryo for transplantation. Prenatal diagnosis was recommended to confirm previous results using Sanger sequencing and karyotyping at an appropriate gestational stage. Tandem mass spectrometry (MS-MS) and gas chromatography-mass spectrometry (GC-MS) were used to detect fetal metabolism after birth. The number of the study cohort is ChiCTR2100053616.ResultsTwo missense variants, c.959G > C (p.Arg320Pro) and c.634G > A (p.Gly212Arg), were validated in the OTC gene in this family. According to the ACMG genetic variation classification criteria, the missense variant c.959G > C can be considered as “pathogenic”, and the missense variant c.634G > A can be regarded as “likely benign.” PGT-M identified a female embryo carrying the heterozygous variant c.959G > C (p.Arg320Pro), which was selected for transplantation. Prenatal diagnosis revealed the same variant in the fetus, and continued pregnancy was recommended. A female baby was born, and her blood amino acid testing and urine organic acid testing were regular. Follow-up was conducted after six months and indicated the girl was healthy.ConclusionOur research first validated the segregation of both c.959G > C and c.634G > A variants in the OTC gene in a Chinese OTCD family. Then, we classified variant c.959G > C as “pathogenic” and variant c.634G > A as “likely benign”, providing corresponding theoretical support for genetic counseling and fertility guidance in this family. PGT-M and prenatal diagnosis were recommended to help the couple receive a female baby successfully with a six-month follow-up.

O-323 Sex-dependent discrepancies in automatic scoring: implications for the performance of embryo evaluation and selection methods

Abstract Study question Are artificial intelligence-based embryo evaluation and selection algorithms equally effective for male and female embryos? Summary answer Compared to female embryos, male embryos exhibited higher automatic scores, superior performance in KIDScore D5 v3, and poorer performance in iDAScore v2 in predicting implantation. What is known already It is known that the secondary sex ratio favors males in assisted reproductive techniques. The reason could be an unbalanced selection, as there is evidence that male embryos undergo faster development and have better morphology than female embryos. Based on this premise, artificial intelligence (AI) models currently used for embryo selection may not perform equally well for embryos of both sexes. We aimed to analyze the performance of two models for assessing male and female embryos: KIDScore D5 v3 and iDAScore v2. Study design, size, duration This is a retrospective cohort study involving 481 patients included in the preimplantation genetic testing program over a year and a half. The euploid embryos (n = 1,181) were divided into male (n = 577) and female (n = 604) groups and the automatic scores received by two artificial intelligence-based evaluation models (KIDScore D5 v3 and iDAScore v2) were considered with respect to implantation. Participants/materials, setting, methods All embryos underwent assisted hatching on day 3 of embryo development and trophoectoderm biopsy at the blastocyst stage (day 5 or 6). The embryos’ sex chromosomes were revealed using a highly validated method. Routine embryo assessment and selection were performed using conventional morphology according to ASEBIR criteria. Retrospectively, embryos were automatically scored from 1 to 9.9. Means, standard deviations, odds ratios (OR), and areas under the ROC curve (AUC) were calculated using IBMSPSS Statistics software. Main results and the role of chance In general, male embryos exhibited a significantly higher iDAScore than female embryos (6.9±2.2 vs. 6.4±2.5)**; no differences for KIDScore (6.1±1.7 vs. 5.9±1.8). This indicates that if the selection were based on the iDAScore, more male embryos would be transferred. Male embryos: 323 male embryos were transferred with an implantation rate of 53.8%. Implanted male embryos had higher automatic score compared to non-implanted ones (6.7±1.6 vs. 6±1.7 for KIDScore** and 7.3±2 vs. 6.8±2.1 for iDAScore*). For KIDScore: OR = 1.3, 95% CI [1.1-1.5] and AUC 0.612 (0.545-0.678). For iDAScore: OR = 1.1, 95% CI [1-1.3] and AUC 0.575 (0.511-0.638). This suggests a higher performance of KIDScore in predicting implantation in male embryos. Female embryos: 318 female embryos were transferred with an implantation rate of 47.7%. Implanted female embryos had higher iDAScore compared to non-implanted ones (7±2.3 vs. 6.3±2.5)*; no differences for KIDScore (6.2±1.8 vs. 6±1.8). For KIDScore: OR = 1.1, 95% CI [0.9-1.2] and AUC 0.542 (0.472-0.612). For iDAScore: OR = 1.1 95% CI [1-1.2] and AUC 0.584 (0.520-0.648). This suggests a higher performance of iDAScore in predicting implantation in female embryos. *pvalue&amp;lt;0.05; **pvalue&amp;lt;0.01 Limitations, reasons for caution The primary limitation of our study is the assisted hatching process performed on embryos on day 3 of development, which may impact late-stage development. This could be a possible reason for the considerably lower performance of the models. Wider implications of the findings Our study revealed objective differences between male and female embryos, as well as distinct performance for two AI-based embryo selection models. These findings suggest that the employed embryo selection model may impact the secondary sex ratio. Trial registration number Not applicable

P-168 Both artificial intelligence and manual embryo selection methods show sex-bias, favouring male embryos – insights from the largest embryo sex study using time-lapse and PGT-A

Abstract Study question Do AI-based and manual embryo selection strategies based on morphological and kinetic evaluation favour male over female embryos? Could the differences enable sex selection? Summary answer Male embryos receive higher-quality scores using both AI-based models and manual grading, but not using deep-learning approaches. The differences, although present, preclude reliable sex selection. What is known already A key aspect of IVF, embryo selection, involves methods executed manually via direct embryo morphological assessment, or by artificial intelligence algorithms which assess parameters such as tPNf, t2, t3, t4, t5, t8, tB and ICM and TE grades using time-lapse imaging. However, all current embryo selection models do not take embryo sex into account despite reported differences in developmental timings between XX and XY embryos. If the predictions of embryo selection algorithms themselves are affected by embryo sex, their accuracy and fairness may be impacted, leading to sex disparities in populations heavily reliant on IVF. Study design, size, duration A retrospective study was conducted on 1411 embryos with known sex information following PGT-A, at a single centre between 2018-202, making this the largest study to date interrogating morphological and morphokinetic sex differences using time-lapse and PGT-A data. Three embryo assessment methods were interrogated: manual morphological grading (Gardener system), KIDScore D3 (VitroLife) and CHLOE (Fairtility). These algorithms are representative of the current embryo grading landscape, encompassing manual selection, traditional machine-learning and modern deep-learning approaches, respectively. Participants/materials, setting, methods Kinetic parameters were annotated using CHLOE on incubator time-lapses. KIDScore computation was implemented according to Petersen et al.,2016 making use of kinetic annotations from CHLOE. Manual morphological grading was done at the blastocyst stage using a modified Gardner system. Mann-Whitney U and chi-squared tests compared XX and XY gradings, with detailed analyses on morphokinetics (tPNf,t2-t9+,cc2,cc3,tM,tSB-tEB,ICM and TE grades). To evaluate the ability of morphokinetic differences to predict embryo sex, four machine learning models were built. Main results and the role of chance Overall, XY embryos (4.182 ± 1.353, N = 692) were more likely to be assigned higher scores than XX embryos (4.022 ± 1.420, N = 642) by the machine learning-based KIDScore (U = 207604, p = 0.0182). Similarly, XY (462/668) blastocysts were more likely to receive good grades than XX (351/614) blastocysts under manual morphological grading (χ2 =19.843, df = 1, p &amp;lt; 0.00001). We pinpoint the source of sex disparities in conventional morphological grading to variations in trophectoderm morphology (male embryos receiving higher TE grades). Nevertheless, the observed differences, although present, were of marginal magnitude, and the overlap between the sexes substantial enough to preclude reliable sex selection. No significant difference was found in the scores assigned by more advanced deep learning methods such as CHLOE EQ scores between XX (0.787 ± 0.276, N = 628) and XY (0.802 ± 0.254, N = 679) embryos (U = 204621, p = 0.208). Limitations, reasons for caution This retrospective, single-centre study encounters limitations such as selection bias and constrained generalisability beyond the specific centre. It focuses on blastocyst-stage sex analysis, which, while statistically-powerful, may not fully represent the societal implications of embryo selection on sex ratios. The use of automated kinetic annotations, while efficient, might introduce inaccuracies. Wider implications of the findings Our findings raise significant questions surrounding the fairness of widely-used embryo assessment methods. We highlight the urgent need for the reproductive health community to recognise and address algorithmic bias, ensuring equitable and ethical treatment of all embryos, especially in the context of increasing global reliance on assisted reproduction. Trial registration number not applicable

P-119 Factors affecting male-to-female ratio at birth in IVF/ICSI cycles

Abstract Study question Are there any factors during an IVF cycle that can influence the ratio of male or female preimplantation embryos? Summary answer Semen preparation technique significantly influences the male-female ratio of newborns through IVF-ICSI with a greater proportion of boys with gradients and girls with swim up. What is known already Although some works have related the fertilization technique or the transfer of blastocysts with the male-female ratio in those born through assisted reproduction, there are limited studies that investigate the relationships of the various factors involved during an IVF/ICSI cycle with the ratio male-female at birth. Furthermore, the number of studies that looked at the effect of embryo morphokinetics in sex ratio at birth, produced contradictory results. Study design, size, duration Retrospective, multicenter study at seven reproductive units belonging to the same center in Spain. The study included 202 patients who underwent IVF/ICSI treatment between January 2022 and December 2022, ensuring that 100% implantation was obtained. All cycles were accomplished with time-lapse, and the transfers were performed at the blastocyst stage. The sex was confirmed at birth. Participants/materials, setting, methods Sex ratios of 223 live births were compared regarding to fertilization method (IVF or ICSI), age, culture medium, embryo morphokinetics (t2, t3, t4,t5,tM,tSB,tBc,cc2,s2), twinship, origin of oocytes and sperm, embryo quality and sperm preparation technique (swim-up vs gradients). The Student’s T test was used for metric variables and the χ2 test for dichotomous variables (p &amp;lt; 0.05). To assess if the rest of the metric variables influenced sex, the Kolmogorov-Smirnov test and the Pearson test were used. Main results and the role of chance 223 live births were obtained: 181 single births after the transfer of one blastocyst and 42 double births of the same sex after the transfer of two blastocysts. Among the 223 newborns included in the study, a statistically significant predominance (p &amp;lt; 0.05) of males was found: 58.3% (95%CI:51.8-64.8%) of boys (n:130) compared to 41.7% (95%CI:35.2-48.2%) of girls (n :93). The mean age of the patients was 38.3 years (SD: 4.87), with no significant differences found regarding the sex ratio, as well as with the fertilization technique (p = 0.937), culture medium (p = 0.096), twinning (p = 0.222), the origin of the oocytes (p:0.608), the origin of the semen (p:0.721) and embryo quality (P:0.186). A statistically significant relationship was found between the sex of the newborn and the semen preparation technique (62.1% of male with gradients versus 32.1% with swim up, p:0.003), which was confirmed using a multivariate binary logistic regression model (p:0.0200). Analyzing each of the parameters obtained from the time lapse with respect to the sex of the newborns in a univariate manner with Student’s T for independent samples no significant differences were found in the division times studied or in the times of the cell cycle and synchrony. Limitations, reasons for caution The main limitation is the necessity of a larger sample size and to further investigate the reasons for these results. Further studies with standardized culture conditions and well-justified inclusion criteria are needed to reach robust and reliable conclusion. Wider implications of the findings While the study needs for more extensive sample size for future research, is the first time to suggest that method of sperm preparation may have an impact on the sex ratio at birth. Understanding which factors within ART process can affect sex ratio remain important considerations. Trial registration number not applicable

P-560 Characterization of ploidy level and parental genome inheritance abnormalities in preimplantation human embryos

Abstract Study question Can preimplantation genetic testing (PGT) platforms with integrated genotyping aid the characterization of genome-wide chromosomal abnormalities in human embryos and factors contributing to their occurrence? Summary answer Signatures detected by genotype-criteria-based models in PGT pinpoint origin of haploidy, triploidy and genome-wide uniparental disomy (gwUPD), highlighting recombination deficiencies potentially underpinning these phenomena. What is known already While there is a growing body of knowledge on the frequency and origin of individual chromosome abnormalities, very little is known about genome-wide chromosomal abnormalities in human development. Indeed, genome-wide abnormalities are incompatible with normal human development, making the preimplantation stage the ideal time to characterize them. Novel PGT technologies provide the research and clinical opportunity to characterize both known ploidy-level abnormalities (triploid/haploid) and identify theoretical genome-wide abnormalities undetectable at later stages of development. Study design, size, duration PGT genotyping data from 96660 embryos were used to assess incidence of ploidy-level abnormalities and correlation with potential predisposing clinical factors. Parental origin of putative haploidy and triploidy was assessed on 14 and 41 embryos with parental genotypes available (trios), respectively. For the 41 triploid trios, meiotic origin and recombination rates were also evaluated. A novel genotype-criteria based model to detect theoretical gwUPD and meiotic origin was developed on a semi-contemporaneous 74009 female embryo dataset. Participants/materials, setting, methods PGT was performed with targeted next-generation sequencing, including 5000 genome-wide SNPs. Informative SNPs from trios were used to identify parental origin of haploidy/triploidy. Informative SNPs within 5cM of the centromere were used to evaluate meiotic origin and extended beyond 5cM to identify maternal recombination observing shifts between single (SPH) and biparental (BPH) homologs. gwUPD was predicted when low genome-wide heterozygosity, increased homozygosity in the centromeric region, and decreased similarity to sibling embryos were observed. Main results and the role of chance Genome wide ploidy abnormalities were detected in 1.1% (n = 1.063/96.660; CI 95%: 1-1,2%) of embryos. Haploidy was mainly of paternal origin (n = 12/14), whereas triploidy was exclusively maternal in origin (n = 41/41) and prominently due to meiosis II errors (n = 30/41; 73.2%; 95% CI:57.1-85.8%). Advanced maternal age was positively associated with an increased risk of triploid conceptions (OR = 1.059 per year; p &amp;lt; 0.001). Investigation of maternal recombination rates detected by three consecutive SNPs shifting between SPH and BPH (with a calculated genotyping error of 0.16%) in triploid embryos revealed an outlier group of 7 embryos with statistically lower (P &amp;lt; 0.001) identified crossover events (0-2 events genome-wide) compared to the average 23.7 detectable events genome-wide (SD:4.3). Recombinant gwUPD was predicted in 41 out of 74009 human embryos (0.05%; 95% CI:0.04-0.07%). These embryos showed a high homozygosity in pericentromeric regions, as a proof of each pair of chromosomes being the same parental homolog resulting from a faulty meiosis II, but with detectable heterozygosity regions throughout the rest of the genome, indicating that crossovers occurred between the two parental homologs during prophase I. Multifocal biopsies from the same embryos confirmed the reproducibility of identification of the number of recombination events in triploid embryos and the gwUPD prediction. Limitations, reasons for caution This assay genotypes only a portion of the genome and underestimation of recombination events is possible. Our current detection of gwUPD is limited to recombinant uniparental isodisomy of MII origin only. Therefore, the exact prevalence of these abnormal gwUPD configurations requires further understanding and analysis. Wider implications of the findings Genotyping in routine PGT can identify severe genome-wide abnormalities with associated gestational complications that would otherwise go undiagnosed. Comprehensive characterization of these abnormalities is not possible at later stages of development. Therefore, PGT offers a unique opportunity to delve into their biological origin and identify novel biomarkers for embryo competence. Trial registration number not applicable

P-033 Microfluific sperm sorting does not impact the primary sex ratio in IVF-ICSI treatments

Abstract Study question Does sperm selection through microfluidics alter the primary sex ratio (PSR) when compared to density gradients? Summary answer The PSR is not modified when employing a microfluidic sperm sorting when comparing to the conventional density gradient technique for sperm selection. What is known already The PSR refers to gender after fertilization, this is often expressed as the male births per female births. Little is known about the sex ratio in natural and assisted reproduction treatments (ART) since it is difficult to establish in ART as it requires preimplantation genetic testing (PGT). Previous studies have been estimating the variation in the PSR based on the fertilization technique used or the day of embryo transfer. Even the influence of sperm selection techniques has not received much attention, using traditional sperm selection methods such as density gradients or swim-up, a PSR of 1.07 has been estimated. Study design, size, duration This is a retrospective study that includes 1764 blastocysts stage embryos biopsied from 522 autologous preimplantation genetic testing for aneuploidies cycles (PGT-A) performed between January of 2020 and December of 2022 and analysed through Next Generation Sequencing (NGS). All the embryos were cultured under the same conditions with one-step media in a time-lapse incubator and the PGT-A was performed in day 5 or day 6 of culture to determine the embryo ploidy status. Participants/materials, setting, methods Two groups were established based on the sperm selection method before ICSI. The study group using a microfluidic system (MS; ZyMōtICSI® or ZyMōtMulti®) included 256 blastocysts from 98 cycles, and a control group using density gradients (DG), with 1058 blastocysts from 424 cycles were compared. PGT-A determined the embryonic sex and for the PSR estimation, number abnormalities in the sexual chromosomes were excluded (i.e. XXY, X0, XYY). Chi-square test was performed to compare the groups. Main results and the role of chance Our data show that there was no statistical significance in the overall ratio of XY:XX embryos biopsied between both groups (p = 0.09). The PSR was 0.89 (47:53) in the study group (MS) and 1.12 (53:47) in the control group (DG). Although not statistically significant, the rate of XX embryos was 6% higher than XY embryos in the MS group. In contrast, in the DG group, the same increase in the rate of XY embryos was observed. If we consider only the euploid embryos which are the potentially born babies, the PSR acquires values of 0.96 for the DG group and 0.92 for the MS group (p = 0.86), showing an increase in euploid female embryos transferred compared to those euploid male embryos. However, it was not statistically significant. Limitations, reasons for caution The performance of PGT-A on blastocysts does not provide an entirely accurate representation of the PSR since it is carried out 5/6 days after the moment of fertilization; nevertheless, it is the technique that comes closest. Also, the low number of blastocysts in the study group represents a limitation. Wider implications of the findings The sperm selection for ICSI with microfluidic technology does not significantly alter the PSR. Trial registration number Not applicable

Non-destructive prediction of fertility and sex in chicken eggs using the short wave near-infrared region

The objective of this study was to evaluate the ability of a handheld near-infrared device (900–1600 nm) to predict fertility and sex (male and female) traits in-ovo. The NIR reflectance spectra of the egg samples were collected on days 0, 7, 14 and 18 of incubation and the data was analysed using principal component analysis (PCA), linear discriminant analysis (LDA) and support vector machines classification (SVM). The overall classification rates for the prediction of fertile and infertile egg samples ranged from 73 % to 84 % and between 93 % to 95 % using LDA and SVM classification, respectively. The highest classification rate was obtained on day 7 of incubation. The classification between male and female embryos achieved lower classification rates, between 62 % and 68 % using LDA and SVM classification, respectively. Although the classification rates for in-ovo sexing obtained in this study are higher than those obtained by chance (50 %), the classification results are currently not sufficient for industrial in-ovo sexing of chicken eggs. These results demonstrated that short wavelengths in the NIR range may be useful to distinguish between fertile and infertile egg samples at days 7 and 14 during incubation.

Best quality vs. sex selection – an analysis of embryo selection preferences for patients undergoing preimplantation genetic testing for aneuploidy over a 10-year period

PurposeInvestigate patient preferences in embryo selection for transfer regarding quality versus sex in IVF/ICSI cycles with PGT-A and assess associated clinical implications.MethodsRetrospective cohort study at a university fertility practice from January 2012 to December 2021. Included were patients undergoing single frozen euploid transfers with at least one embryo of each sex available. Primary outcomes were preference for embryo selection (quality vs. sex) and sex preference (male vs. female). Trends over 10 years were evaluated and clinical outcomes, including clinical pregnancy rate (CPR), sustained implantation rate (SIR), and live birth rate (LBR), were compared.ResultsA total of 5,145 embryo transfer cycles were included; 54.5% chose the best-quality embryo, while 45.5% selected based on sex. Among those choosing based on sex, 56.5% chose male embryos and 43.5% chose female. Preference for quality remained consistent over the decade (p = 0.30), while male embryos were consistently favored (p = 0.64). Best-quality embryos had higher grades (p < 0.001). Clinical outcomes were similar between groups (CPR: 74.4% vs. 71.9%, p = 0.05; SIR: 64.9% vs. 63.4%, p = 0.26; LBR: 58.8% vs. 56.7%, p = 0.13), and between male and female embryo selections.ConclusionsSex selection remains common, with 45.5% selecting embryos based on sex, predominantly favoring males. This trend persisted over 10 years, with comparable clinical outcomes regardless of selection criteria.

Sex- and caste-specific developmental responses to juvenile hormone in an ant with maternal caste determination.

Juvenile hormone is considered to be a master regulator of polyphenism in social insects. In the ant Cardiocondyla obscurior, whether a female egg develops into a queen or a worker is determined maternally and caste-specific differentiation occurs in embryos, so that queens and workers can be distinguished in a non-invasive manner from late embryogenesis onwards. This ant also exhibits two male morphs - winged and wingless males. Here, we used topical treatment with juvenile hormone III and its synthetic analogue methoprene, a method that influences caste determination and differentiation in some ant species, to investigate whether hormone manipulation affects the development and growth of male, queen- and worker-destined embryos and larvae. We found no effect of hormone treatment on female caste ratios or body sizes in any of the treated stages, even though individuals reacted to heightened hormone availability with increased expression of krüppel-homolog 1, a conserved JH first-response gene. In contrast, hormone treatment resulted in the emergence of significantly larger males, although male morph fate was not affected. These results show that in C. obscurior, maternal caste determination leads to irreversible and highly canalized caste-specific development and growth.

XIST dampens X chromosome activity in a SPEN-dependent manner during early human development.

XIST (X-inactive specific transcript) long noncoding RNA (lncRNA) is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female preimplantation embryos without triggering X chromosome silencing. The XACT (X-active coating transcript) lncRNA coaccumulates with XIST on active X chromosomes and may antagonize XIST function. Here, we used human embryonic stem cells in a naive state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during preimplantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and dampens the transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional before XCI, confirms the existence of a transient process of X chromosome dosage compensation and reveals that XCI and dampening rely on the same set of factors.