Embryo Sex Research Articles

Variation in thermal traits describing sex determination and development in Western Australian sea turtle populations

Abstract Both the development rate and sex of sea turtle embryos depend on incubation temperature, as all species of sea turtle are ectotherms and show temperature‐dependent sex determination (TSD). Theory predicts that selection should act on populations to optimize developmental times and primary sex ratios. In this study, we use a consistent methodology to measure development rates and model the reaction norm that defines TSD in three populations of flatback turtles Natator depressus and two populations of green turtles Chelonia mydas that nest in Western Australia. We show that development rates vary between and within species, likely reflecting adaptation to local beach microclimates. Similarly, the parameters that define the TSD reaction norm vary between the two species, and among N. depressus populations, with pivotal temperatures (TPIV) varying by 1.5°C (29.6–31.1°C), and the transitional ranges of temperatures (TRT) varying by 1.4–3.3°C. In contrast, we found a similar TPIV for the two C. mydas populations, but a wider TRT at the northernmost tropical rookery. Our findings support the view that thermal parameters in geographically separated populations of sea turtles are broadly similar, but the variation we describe will be highly relevant for predicting how populations will respond to climate change. A free Plain Language Summary can be found within the Supporting Information of this article.

Could PGT-A pick up true abnormalities that have clinical relevance? Retrospective analysis of 1043 embryos

ObjectiveTo determine whether preimplantation genetic testing for aneuploidy (PGT-A) could pick up true abnormalities that have clinical relevance. Materials and methodsThis was a retrospective cohort study of patients who underwent in vitro fertilization with PGT-A from 2015 to 2017. We evaluated the associations of aneuploidy and mosaicism with maternal age, the chromosome abnormalities present in individual chromosomes, and the effect of embryo sex on the proportion of each type of error in the four chromosomes most frequently affected. Result(s)A total of 1043 embryos from 255 patients (mean maternal age = 39 ± 4 years) were included in the initial analysis. Of these, 36% (377/1043) were euploid, 47% (487/1043) were aneuploid, 13% (140/1043) contained mosaicism, and 4% (39/1043) gave no result. We excluded the 39 embryos with no result; thus, 1004 embryos were included in the analysis. Increased aneuploidy was associated with increased maternal age, but the rate of embryo mosaicism was not. A combined analysis of aneuploidy with noncomplex abnormalities and mosaicism showed that chromosomes 22, 21, 16, and 15 were the most frequently involved. Chromosome 22 showed the highest proportion of mosaicism and chromosome 15 showed the highest proportion of aneuploidy. When we included embryo sex in the analysis, embryo sex was associated with these chromosome errors in the most susceptible chromosome, 22. Conclusion(s)PGT-A showed that chromosomes 22, 21, 16, and 15 were the most frequently involved among common chromosome abnormalities, comparable with those of published data analyzed from spontaneous abortion. This result suggested that PGT-A could pick up abnormalities that have clinical relevance to spontaneous abortion. Moreover, we identified a role of embryo sex in these chromosomal errors on chromosome 22.

Noninvasive sexing of human preimplantation embryos using RT-PCR in the spent culture media: A proof-of-concept study

Preimplantation genetic testing (PGT) routinely requires biopsy which is an invasive approach. The aim of this study was to examine a noninvasive approach for sexing of preimplantation embryos using polymerase chain reaction (PCR)/reverse transcriptase-PCR (RT-PCR) based on the presence of SRY DNA/RNA in the spent culture medium.Two groups were evaluated: in group 1, 40 embryos of routine PGT volunteers were cultured individually after biopsy and in group 2, 31 embryos were cultured individually until Day-5 post-fertilization. Each group was further divided into three subgroups: RNA extraction (RE), nucleic acid (NA) and DNase treated (DT). In the NA and DT subgroups, cDNA synthesis was performed directly on culture medium with or without DNase treatment in DT and NA subgroups, respectively. The results of sexing based on the PCR/RT-PCR in the culture medium, were compared with the results of sexing by fluorescence in situ hybridization (FISH) technique.In group 1, all samples were correctly diagnosed. In group 2, five female samples were misdiagnosed. Test’s sensitivity, specificity and accuracy were 100 %, 94.44 % and 96.88 %, in RE, 100 %, 81.82 % and 93.55 % in DT and 100 %, 71.43 % and 85.71 % in NA, respectively.Preimplantation sexing without embryo biopsy, in the spent embryo culture media using RNA amplification based methods including RE and DT seem to be more reliable while nucleic acid based method, NA, led to the highest misdiagnoses probably due to DNA contamination. Since all male samples were correctly diagnosed in all subgroups of this preliminary study, preimplantation noninvasive sexing on culture medium seems feasible, however all sources of nucleic acid contamination must be carefully avoided.

Sexing of pre-implantation ovine embryos through polymerase chain reaction-based amplification of GAPDH, SRY and AMEL genes.

The ability to identify the sex of embryo and control of sex ratio has a great commercial importance to livestock industry. Prediction of embryonic sex could be useful in the management decisions of sex selection in breeding programs. Several methods have been attempted to determine the sex but the polymerase chain reaction (PCR)-based sexing method is generally favoured, as it is cost effective, simple and reliable. The aim of the present study was to identify sex of sheep embryos produced in vitro through amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sex-determining region Y (SRY) and amelogenin genes present in genomic DNA (gDNA) of embryos through PCR. To avoid false interpretation of the result by no amplification of SRY in female embryos, a duplex PCR was approached to amplify combinedly SRY and GAPDH genes. Sex-specific blood was used in PCR as positive control. In vitro sheep embryos were produced as per standardized protocol of laboratory. Sexing of sex-specific blood and in vitro produced embryos were approached though PCR to amplify the respective genes using gDNA present in the sample without its traditional isolation. The accuracy of sex prediction for embryos was 100% by this procedure.

Generation of Transgenic Cloned Buffalo Embryos Harboring the EGFP Gene in the Y Chromosome Using CRISPR/Cas9-Mediated Targeted Integration.

Sex control technology is of great significance in the production of domestic animals, especially for rapidly breeding water buffalo (bubalus bubalis), which served as a research model in the present study. We have confirmed that a fluorescence protein integrated into the Y chromosome is fit for sexing pre-implantation embryos in the mouse. Firstly, we optimized the efficiency of targeted integration of exogenous gene encoding enhanced green fluorescent protein (eGFP) and mCherry in Neuro-2a cells, mouse embryonic stem cells, mouse embryonic cells (NIH3T3), buffalo fetal fibroblast (BFF) cells. The results showed that a homology arm length of 800 bp on both sides of the target is more efficient that 300 bp or 300 bp/800 bp. Homology-directed repair (HDR)-mediated knock-in in BFF cells was also significantly improved when cells were supplemented with pifithrin-μ, which is a small molecule that inhibits the binding of p53 to mitochondria. Three pulses at 250 V resulted in the most efficient electroporation in BFF cells and 1.5 μg/mL puromycin was found to be the optimal concentration for screening. Moreover, Y-Chr-eGFP transgenic BFF cells and cloned buffalo embryos were successfully generated using CRISPR/Cas9-mediated gene editing combined with the somatic cell nuclear transfer (SCNT) technique. At passage numbers 6–8, the growth rate and cell proliferation rate were significantly lower in Y-Chr-eGFP transgenic than in non-transgenic BFF cells; the expression levels of the methylation-related genes DNMT1 and DNMT3a were similar; however, the expression levels of the acetylation-related genes HDAC1, HDAC2, and HDAC3 were significantly higher (p < 0.05) in Y-Chr-eGFP transgenic BFF cells compared with non-transgenic cells. Y-Chr-eGFP transgenic BFFs were used as donors for SCNT, the results showed that eGFP reporter is suitable for the visualization of the sex of embryos. The blastocyst rates of cloned buffalo embryos were similar; however, the cleavage rates of transgenic cloned embryos were significantly lower compared with control. In summary, we optimized the protocol for generating transgenic BFF cells and successfully generated Y-Chr-eGFP transgenic embryos using these cells as donors.

Maternal effects and urbanization: Variation of yolk androgens and immunoglobulin in city and forest blackbirds.

Wildlife inhabiting urban environments exhibit drastic changes in morphology, physiology, and behavior. It has often been argued that these phenotypic responses could be the result of micro‐evolutionary changes following the urbanization process. However, other mechanisms such as phenotypic plasticity, maternal effects, and developmental plasticity could be involved as well. To address maternal effects as potential mechanisms, we compared maternal hormone and antibody concentrations in eggs between city and forest populations of European blackbirds (Turdus merula), a widely distributed species for which previous research demonstrated differences in behavioral and physiological traits. We measured egg and yolk mass, yolk concentrations of androgens (androstenedione [A4], testosterone [T], 5α‐dihydrotestosterone [5α‐DHT], and immunoglobulins [IgY]) and related them to population, clutch size, laying order, embryo sex, and progress of breeding season. We show (a) earlier onset of laying in the city than forest population, but similar egg and clutch size; (b) higher overall yolk androgen concentrations in the forest than the city population (sex‐dependent for T); (c) greater among‐female variation of yolk T and 5α‐DHT concentrations in the forest than city population, but similar within‐clutch variation; (d) similar IgY concentrations with a seasonal decline in both populations; and (e) population‐specific positive (city) or negative (forest) association of yolk A4 and T with IgY concentrations. Our results are consistent with the hypotheses that hormone‐mediated maternal effects contribute to differences in behavioral and physiological traits between city and forest individuals and that yolk androgen and immunoglobulin levels can exhibit population‐specific relationships rather than trade‐off against each other.

Sex Determination of in Vitro Produced Goat Embryos by Epididymal Spermatozoa Selected by Swim up Technique

The study was conducted to determine the sex of the male and female goat embryos produced in vitro fertilization by Epididymal spermatozoa of local buck and separated by swim-up technique. The sex of produced embryo was identified by polymerase chain reaction (PCR). Results indicate that the fertilization by Epididymal spermatozoa selected using Swim-up technique at 200×g centrifugation showed that 48.70 ±0.62 % of spermatozoa were detected in the supernatant while the precipitate contained 42.1±0.70. Spermatozoa in the supernatant were used for IVF of matured oocytes. The sex of goat embryos produced in vitro fertilization was determined by polymerase chain reaction (PCR) using specific primers to detect the SRY gene. The percentage of male embryos recorded 71.73% while female embryos recorded only 28.26% from the total goat embryos obtained after IVF by sperms selected using swim-up at centrifugation force of 200×g. It was concluded that the use of swim-up technique up at centrifugation force of 200×g on Epididymal spermatozoa showed the ability of selection male embryos in caprine.

Targeting of the Dosage-Compensated Male X-Chromosome during Early Drosophila Development

SUMMARYDosage compensation, which corrects for the imbalance in X-linked gene expression between XX females and XY males, represents a model for how genes are targeted for coordinated regulation. However, the mechanism by which dosage compensation complexes identify the X chromosome during early development remains unknown because of the difficulty of sexing embryos before zygotic transcription using X- or Y-linked reporter transgenes. We used meiotic drive to sex Drosophila embryos before zygotic transcription and ChIP-seq to measure the dynamics of dosage compensation factor targeting. The Drosophila male-specific lethal dosage compensation complex (MSLc) requires the ubiquitous zinc-finger protein chromatin-linked adaptor for MSL proteins (CLAMP) to identify the X chromosome. We observe a multi-stage process in which MSLc first identifies CLAMP binding sites throughout the genome, followed by concentration at the strongest X-linked MSLc sites. We provide insight into the dynamics of binding site recognition by a large transcription complex during early development.

Sex affects immunolabeling for histone 3 K27me3 in the trophectoderm of the bovine blastocyst but not labeling for histone 3 K18ac.

The mammalian embryo displays sexual dimorphism in the preimplantation period. Moreover, competence of the embryo to develop is dependent on the sire from which the embryo is derived and can be modified by embryokines produced by the endometrium such as colony stimulating factor 2 (CSF2). The preimplantation period is characterized by large changes in epigenetic modifications of DNA and histones. It is possible, therefore, that effects of sex, sire, and embryo regulatory molecules are mediated by changes in epigenetic modifications. Here it was tested whether global levels of two histone modifications in the trophectoderm of the bovine blastocyst were affected by sex, sire, and CSF2. It was found that amounts of immunolabeled H3K27me3 were greater (P = 0.030) for male embryos than female embryos. Additionally, labeling for H3K27me3 and H3K18ac depended upon the bull from which embryos were derived. Although CSF2 reduced the proportion of embryos developing to the blastocyst, there was no effect of CSF2 on labeling for H3K27me3 or H3K18ac. Results indicate that the blastocyst trophoctoderm can be modified epigenetically by embryo sex and paternal inheritance through alterations in histone epigenetic marks.

Identification of the Sex of Pre-implantation Mouse Embryos Using a Marked Y Chromosome and CRISPR/Cas9

Although numerous attempts have been made to alter the sex ratio of the progeny of mammals, the limitations of current technologies have prevented their widespread use in farm animals. The presence or absence of a Y chromosome determines whether a mammalian embryo develops as a male or female, and non-invasive genetic reporters such as fluorescence protein markers have been intensively applied in a variety of fields of research. To develop a non-invasive and instantaneous method for advance determination of the sex of embryos, we developed a Y chromosome-linked eGFP mouse line that stably expresses green fluorescent protein under the control of the CAG promoter. The development of the CRISPR/Cas9 system has made it easy to deliver an exogenous gene to a specific locus of a genome, and linking a tracer to the Y chromosome has simplified the process of predicting the sex of embryos collected by mating a Y-Chr-eGFP transgenic male with a wild-type female. XY embryos appeared green, under a fluorescence microscope, and XX embryos did not. Y chromosome-linked genes were amplified by nested PCR to further confirm the accuracy of this method, and the simultaneous transplantation of green and non-green embryos into foster mothers indicated that 100% accuracy was achieved by this method. Thus, the Y-Chr-eGFP mouse line provides an expeditious and accurate approach for sexing pre-implantation embryos and can be efficiently used for the pre-selection of sex.

The relationship between male social status, ejaculate and circulating testosterone concentration and female yolk androgen transfer in red junglefowl (Gallus gallus)

Several studies show that avian females prefer males based on their secondary sexual ornaments and dominance status. We tested in red junglefowl (Gallus gallus) how comb size affected the result of fighting and how the dominance status related to testosterone concentrations in their circulation and ejaculates. We subsequently tested how social status was related to female reproductive investment, including yolk hormone transfer. We found that after a fight 1) winners increased plasma T and decreased ejaculates T whereas losers' T remained unchanged, and 2) plasma T of winners was higher but ejaculates T was lower than those of losers. We argued those are consistent with the different reproductive strategies of dominant and subordinate males. Furthermore, in line with offspring sex-dependent growth patterns females transferred significantly more androstenedione to female than male embryos when mated with winners, while doing the opposite when mated with losers. We concluded therefore that female reproductive investment was affected by both partner quality and embryo sex. The results indicate that male quality influences sex-specific maternal investment, which could be mediated by ejaculate testosterone concentration.

Oil dispersant Corexit 9500 is weakly estrogenic, but does not skew the sex ratio in Alligator mississippiensis.

During the Deepwater Horizon oil spill, vast quantities of a chemical dispersant Corexit 9500 were applied in remediation efforts. In addition to the acute toxicity, it is essential to evaluate Corexit further with a broader scope of long-term sublethal endocrine endpoints. The American alligator (Alligator mississippiensis) is an excellent organism for such an endeavor. It exhibits temperature-dependent sex determination, in which egg incubation temperatures during a thermosensitive period (TSP) in embryonic development determine the sex of embryos. Estrogen signals play a critical role in this process. For example, a single exposure to exogenous estrogen during the TSP overrides the effects of temperature and leads to skewed sex ratios. At a concentration of 100ppm, Corexit significantly induced transcriptional activity of both alligator nuclear estrogen receptors 1 and 2 in vitro in reporter gene assays. To investigate the estrogenic effects of Corexit on gonadal development, alligator eggs were exposed to Corexit at environmentally relevant concentrations (0.25, 2.5 and 25ppm) before the TSP in ovo. Exposure to Corexit at 0.25 and 25ppm significantly delayed hatching and growth. Corexit exposure at any treatment level did not affect sex ratios or testicular mRNA abundance as measured at 1-week post-hatching, suggesting that the combination of Corexit components did not synergize enough to induce ovarian development in ovo. These results point to a need for further investigations on individual and combined components of Corexit to understand better their long-term effects on the development and reproductive health of alligators and other coastal aquatic wildlife.

Global transcriptomic response of bovine endometrium to blastocyst-stage embryos.

The aims of this study were (i) to investigate changes in the global transcriptome of bovine endometrial explants induced by exposure to blastocysts, (ii) to investigate if male and female blastocysts elicit a differential response in the endometrial transcriptome in vitro and (iii) to determine whether bovine endometrium responds to the presence of murine embryos. In Experiment 1, endometrial explants from the same uterus were cultured for 6 h with or without 20 in vitro-produced bovine blastocysts. In Experiment 2, endometrial explants were cultured with male or female bovine blastocysts produced in vitro by IVF either using sex-sorted semen or conventional unsorted semen followed by embryo sexing based on a biopsy. In Experiment 3, endometrial explants were cultured alone or in the presence of bovine blastocysts (n = 25) or murine blastocysts (n = 25). Following culture, explants were snap frozen and stored at -80°C until RNA extraction, qPCR or RNA-Seq. Culture with bovine blastocysts increased endometrial expression of 40 transcripts, all of which were interferon-tau induced. Culture with male or female bovine blastocysts increased transcript abundance of five classic interferon-stimulated genes (MX1, MX2, ISG15, OASY1, RSAD2) in explants; however, there was no difference in abundance of transcripts previously reported to be related to embryonic sex (IFNAR1, IFNAR2, CTGF, ARTN, SLC2A1, SLC2A5). Exposure to murine blastocysts did not elicit any detectable change in transcript abundance. These findings, coupled with our previous data, indicate that very local, interferon-tau-induced changes in endometrial gene expression occur in response to blastocysts; whether such changes play any role in subsequent pregnancy recognition remains to be established.

Relationship of embryo sex to embryo quality, day of blastocyst transformation, and IVF outcomes

Relationship of embryo sex to embryo quality, day of blastocyst transformation, and IVF outcomes

Differential transcriptome dynamics during the onset of conceptus elongation and between female and male porcine embryos

BackgroundPorcine embryos undergo rapid differentiation and expansion between Days 8 and 12 before attaching to the maternal uterine epithelial surface after Day 13. It is known that maternal recognition of pregnancy and successful implantation are driven by mutual interactions between the elongated conceptus and the maternal endometrium. While most of the genes involved in regulation of embryo development are located on autosomal chromosomes, gene expression on sex chromosomes is modulating development through sex-specific transcription. To gain more insights into the dynamic transcriptome of preimplantation embryos at the onset of elongation and into X-linked gene expression, RNA-seq analyses were performed for single female and male porcine embryos collected on Days 8, 10, and 12 of pregnancy.ResultsA high number of genes were differentially expressed across the developmental stages (2174 and 3275 for Days 8 vs 10, and 10 vs 12, respectively). The majority of differentially expressed genes (DEGs) were involved in embryo elongation, development, and embryo-maternal interaction. Interestingly, a number of DEGs was found with respect to embryo sex (137, 37, and 56 on Days 8, 10 and 12, respectively). At Day 8, most of these DEGs were X-linked (96). Strikingly, the number of DEGs encoded on the X chromosome dramatically decreased from Day 10 to Day 12.ConclusionsThe obtained results deepen the understanding about temporary transcriptomic changes in porcine embryos during the phase of conceptus elongation, meanwhile reveal dynamic compensation of X chromosome in the female and distinct transcriptional differences between female and male embryos.

Effect of incubation temperature on sex-dependent embryo mortality and morphological traits in Mallard.

Although birds have genetically determined sex, the sex ratio has been reported to deviate from parity in several studies. Temperature-dependent sex determination, which is common in reptiles, is absent in birds. However, females are able to adjust their investment into eggs according to the sex of the embryo, which may cause sex-specific embryonic mortality. Incubation temperature may also cause sex-biased embryonic mortality, and it may differentially affect the phenotype of male and female hatchlings. We aimed to investigate differences between male and female Mallard embryos regarding their egg size, mortality during incubation and hatchling phenotype in relation to incubation temperature. Mallard eggs were incubated under six constant incubation temperatures (ranging from 35.0 to 38.0 °C). Hatchlings were weighed, and their morphological traits were measured. We determined the sex of hatchlings and unhatched embryos by genetic analysis and found higher male embryonic mortality at 35.5 °C (44 males vs. 28 females) and a higher proportion of female hatchlings at 38 °C (24 males vs. 38 females); however, these results were not statistically significant. Our results suggest that Mallard females do not differentiate quantitatively between sexes during egg production. Male hatchlings were significantly larger but not heavier than females. The size difference between sexes was most pronounced at temperatures around 36 °C, which is the mean temperature of naturally incubated Mallard eggs.

Effect of sex on cryotolerance of bovine embryos produced in vitro

Male and female embryos are known to be different in developmental kinetics, metabolism, gene expression, and epigenetic patterns. Therefore, the objective of this study was to clarify whether the morphological criteria used to select embryos for cryopreservation lead to a deviation in the male:female ratio, and whether vitrification effects vary according to embryo sex. Initially, five sires were tested to evaluate the effect of the bull on embryo development, sex ratio, speed of development, and response to cryopreservation. Results showed that bulls affected (P < 0.05) embryo production, response to cryopreservation, and sex ratio. Then, one bull was selected, and used to produce embryos in vitro to characterize the responses of male and female embryos to vitrification. Results suggested that male and female embryos have the same morphological responses to vitrification, as no differences (P > 0.05) were observed between the two sexes in post-warming survival and re-expansion rates. However, their molecular responses as evaluated by gene expression (FOSL1, HSPB1, CASP3, CASP8, HSPA5, HSPA1A, G6PD, and PGK1) analysis indicated an effect of sex on vitrification; vitrified female embryos exhibited higher mRNA levels of HSPA1A, CASP3, and G6PD compared to their male counterparts. In conclusion, bulls affected embryo production, speed of development, sex ratio, and response to cryopreservation. Male and female embryos differed in their molecular responses to vitrification; and also, deviations in the male:female ratio when selecting embryos for cryopreservation were confirmed.

Identification of sex using SBNO1 gene in the Chinese softshell turtle, Pelodiscus sinensis (Trionychidae)

The Chinese softshell turtle exhibits ZZ/ZW sex determination. To identify the sex of embryos, juvenile and adult individuals, we designed two pairs of polymerase chain reaction primers, SB1-196, which amplifies a fragment of 196 bp in the female and the other, CK1-482, which amplifies the 482-bp fragment in both the sexes. It is validated in 24 adult turtles of known sex, sampled from three different locations. This one-step sexing technique is rapid and easy to perform and is reported for the first time.

Reproductive challenges facing the cattle industry at the beginning of the 21st century

The aim of this review is to pinpoint the areas that require further research for greatest impact to improve the efficiency of dairy and beef production. Increased knowledge about the principal causes of reduced fertility is essential. Increases in milk yield have been at the expense of reduced fertility in dairy cows and although diet has a major impact, the precise interaction between nutrition and reproduction still needs to be characterized in both beef and dairy cows. Furthermore, during periods of inadequate nutrition or stress, the intensity of oestrus is reduced by inadequate exposure to oestradiol. However, it is still unclear which pheromones are involved at oestrus, how synthesis is controlled and how pheromones are detected in herd-mates. GnRH may be involved in behaviour but the brain centres that translate hormonal messages are unknown. Attempts to overcome poor oestrous detection include measurements of milk progesterone, telemetric pressure detectors and devices that record the extra activity at oestrus. Substitution of heat detection by 'hormone treatment remedies' has met strong consumer resistance in Europe. The creation of larger cattle units and increased movements world-wide render herds more susceptible to infectious agents, such as Neospora, Leptospira, Trypanosoma and Bovine Viral Diarrhoea virus (BVDv), but it is unclear how other clinical conditions, such as lameness or endometritis, also interfere with ovarian function. The future of dominant follicles selected within 14 days after parturition is crucial--normal ovulation, prolonged persistence or atresia. Calving carries the greatest risk in the reproductive life of a cow and yet little work has focused on reducing the frequency of this event. For dairy cows, a greater understanding about induced or extended lactation is required. For beef animals, precise induction of twinning and nutritional adjustments could produce two offspring per pregnancy. At the start of pregnancy, the trophoblast produces interferon to prevent luteolysis, but the immunological implications are unknown and it is not clear how the rest of pregnancy is maintained. Profiles of pregnancy specific protein B (PSPB) have increased understanding of embryonic death. However, 25% of cows in abattoirs are pregnant, even though 30% of involuntary cullings are 'for failing to conceive'. Clearly, this is an area of wastage that requires urgent resolution. It is unknown why undernutrition at the time of insemination or in early pregnancy leads to delayed births, low fetal weights and later adverse health. At the end of pregnancy, the fetus controls the onset of parturition, but very little is known about the biochemical control of cervical dilation and placental separation. On the male side, bulls are selected for optimal freezability of semen; however, there is as yet no reliable predictor for semen fertility. Methods for accurately pre-determining the sex of both semen and embryos will revolutionize the dairy and beef industries.

Non-invasive preimplantationsexingusingRT-PCRon the spent embryo culture media

Introduction Preimplantation sexing is an alternative method for Preimplantation GeneticTesting (PGT) to prevent the birth of children suffering from x-linked genetic disorders.Social sexing for non-medical reasons is also legal for some cases. PGT routinely needsto biopsy of embryonic cells or oocyte polar bodies which are invasive and could lead to implantation failure. SRY gene expression begins in male embryos following genome activation in preimplantation embryos. The aim of this study was sexing of preimplantation embryos without biopsy based on the presence of SRY RNA and DNA in the spent culture medium as a biomarker of sexing of human preimplantation embryos. Materials and Methods In this double-blind study, two groups were evaluated. In the first group, on the third day after the biopsy, the human embryos were cultured individually; we received the spent media of the non-degenerated 5-day embryos those have been underwent sexing by Fluorescent In Situ Hybridization(FISH) method via blastomere biopsy on the third day. In the second group, embryos of ART candidateswere cultivated on the third day individuallyuntil development to blastocyst stage; this group of embryos was not biopsied but the blastocysts were fixed for sexing by FISH. Each group was divided to 2 subgroups; in the first subgroup we extracted the RNA from the spent culture medium of each embryo. Following RNA extraction, the total RNA was immediately reverse transcribed to cDNA. In the second subgroup, PCR was performed directly on the culture medium. PCR was performed for SRY and GAPDH genes. The SRY positive embryos were considered as male embryos and those were GAPDH positive and SRY negative considered as females. The results of sexing based on spent culture media was compared with the results of sexing based on FISH. Results For the first group, 12 samples were evaluated, after comparing the results of PCR and FISH, the results for all the samples were concordant.In the second group, we were able to correctly diagnose 12 of the 14 samples. Two embryos with false diagnosis were females that diagnosed as males by direct PCR. Conclusion Preimplantation sexing without embryo biopsy by RT-PCR on the spent embryo culture media seems to be a reliable tool for non-invasive preimplantation sexing. However, direct PCR-based diagnosis might be lead to false results; this is probably due to DNA contamination.