Female Preimplantation Embryos Research Articles

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 < 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 < 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

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.

P-135 No difference in morphokinetic patterns between male and female preimplantation embryos

Abstract Study question Do morphokinetic parameters vary between male and female preimplantation embryos? Summary answer We observed no significant differences in morphokinetic characteristics between male and female preimplantation embryos What is known already Studies across various mammalian species, including humans, suggest that male and female embryos differ in their gene expression and metabolic phenotypes. These variations may inherently affect the timing and duration of key preimplantation events. In the context of ART, such developmental differences may ultimately bias embryo selection, leading to an unbalanced sex ratio in the resulting offspring. To date, only a few studies have explored the relationship between morphokinetic patterns and human embryo sex. While some suggest subtle sex-specific variations in human embryo growth dynamics, results have been conflicting and currently no clear consensus has been reached. Study design, size, duration Retrospective study of morphokinetic data obtained from 175 preimplantation embryos. The study included 175 cycles performed at two IVF centers, between March 2018 and June 2021. Only fresh oocyte donation cycles using intracytoplasmic sperm injection (ICSI) and fresh single embryo transfer (SET) were assessed. Sex of the embryos was obtained from live birth data. Overall, our analysis included 92 female and 83 male embryos. Participants/materials, setting, methods All embryos were cultured in a timelapse incubator in standardized conditions. The morphokinetic parameters assessed included: time to pronuclear fading (tPNf), times to 2–5 cells (t2, t3, t4, t5), time to 8 cells (t8), time to start of blastulation (tSB) and time to full blastocyst stage (tB). All parameters were measured in hours post insemination (hpi). A two-tailed Student’s t-test was used to compare morphokinetics between embryo sexes. A p<0.05 was considered statistically significant. Main results and the role of chance We observed no significant differences in morphokinetic parameters when comparing cycles resulting in female vs. male live births: tPNf (21.8 ± 3.3 vs. 22.3 ± 3.4 hpi; p > 0.39); t2 (24.6 ± 2.5 vs. 25.0 ± 2.5 hpi; p > 0.34); t3 (35.3 ± 3.3 vs. 35.8 ± 3.1 hpi; p > 0.28); t4 (36.3 ± 3.4 vs. 36.9 ± 3.7 hpi; p > 0.20); t5 (47.9 ± 4.7 vs. 48.0 ± 4.8 hpi; p > 0.88); t8 (54.0 ± 6.5 vs. 54.1 ± 6.5 hpi; p > 0.91); tSB (86.3 ± 14.6 vs 85.7 ± 15.5 hpi; p > 0.78) and tB (93.0 ± 16.9 vs. 93.2 ± 17.2 hpi; p > 0.94). These findings suggest that the timing of key developmental events does not vary between male and female ICSI-derived preimplantation embryos. Limitations, reasons for caution The main limitation of this study is its retrospective nature and sample size. We analyzed the data of embryos leading to a live birth; therefore, caution is warranted when generalizing results to non-implanting embryos. Wider implications of the findings Male and female human embryos display similar morphokinetic patterns throughout preimplantation development in our study. Accordingly, embryo selection in a timelapse setting based on developmental characteristics does not bias sex ratios in resulting live births. Trial registration number not applicable

No difference in morphokinetics between male and female preimplantation embryos from ART

Research questionDo morphokinetic parameters vary between male and female preimplantation embryos? DesignThis was a retrospective cohort study of 175 cycles between March 2018 and June 2021 at two reproductive centres. It included time-lapse data from 92 female and 83 male preimplantation embryos exclusively issued from fresh oocyte donation and undergoing intracytoplasmic sperm injection (ICSI). Only fresh elective single-embryo transfers on day 5 were assessed, and the sex of the embryo was confirmed at birth. The morphokinetic parameters analysed were measured in hours post-insemination (hpi). A two-tailed Student's t-test was used to compare the morphokinetics between embryo sexes and a value of P < 0.05 was considered statistically significant. ResultsFollowing strict inclusion criteria to avoid poor-quality preimplantation embryos, no significant differences were found in morphokinetic parameters when comparing cycles that resulted in female versus male live births for the following: time to pronuclear fading (22.1 ± 2.4 versus 22.4 ± 2.9 hpi; P = 0.52); time to the 2-cell stage (24.6 ± 2.5 versus 25.0 ± 2.5 hpi; P = 0.34); time to the 3-cell stage (35.3 ± 3.3 versus 35.8 ± 3.1 hpi; P = 0.28); time to the 4-cell stage (36.3 ± 3.4 versus 36.9 ± 3.7 hpi; P = 0.20); time to the 5-cell stage (47.9 ± 4.6 versus 48.0 ± 4.8 hpi; P = 0.88); time to the 8-cell stage (54.0 ± 6.5 versus 54.1 ± 6.5 hpi; P = 0.91); time to the start of blastulation (86.3 ± 14.6 versus 85.7 ± 15.5 hpi; P = 0.78); and time to the full blastocyst stage (93.0 ± 16.9 versus 93.2 ± 17.2 hpi; P = 0.94). ConclusionsThere are no significant differences in morphokinetics between male and female preimplantation embryos.

P–137 Male embryos take longer to develop to the blastocysts stage

Abstract Study question Are there any differences in developmental timings between male and female preimplantation embryos? Summary answer There is a tendency for statistical difference in the time to reach blastocyst stage for male embryos compared to female embryos What is known already Differences in gene expression and metabolic uptake between male and female preimplantation embryos have been found in animal models and humans. These differences could affect the developmental timings of embryos resulting in differences in either sex. Morphokinetic parameters can precisely assess developmental timings. Only a few studies have analyzed morphokinetic parameters between male and female preimplantation embryos and no consensus has been reached on whether there is any sex-specific difference. The objective of this study is to compare morphokinetic parameters between male and female preimplantation embryos to determine any sex-specific developmental differences. Study design, size, duration This is a retrospective study including 102 preimplantation embryos from February 2018 to February 2020. The morphokinetic parameters obtained from time-lapse records of each embryo were: time to pronuclear fading (tPNf), times to 2–8 cells (t2, t3, t4, t5, t6, t7, t8), time to start of blastulation (tSB) and time to full blastocyst stage (tB). A two-tailed Student’s t-test was used to compare morphokinetic parameters between embryo sexes. A p &amp;lt; 0.05 was considered statistically significant. Participants/materials, setting, methods The study included retrospective time-lapse data from preimplantation embryos giving rise to 51 baby boys and 51 baby girls, as seen at birth. This is a single-center study with standardized culture conditions. Embryos in both study groups issued from cycles with donated oocytes. Only elective blastocyst stage single-embryo transfers (SET) on day 5 were assessed. Main results and the role of chance A tendency to statistical difference (p = [0.1–0.05]) was observed for blastocyst-related morphokinetic parameters: tSB (mean time was 89.6±6.3 hours in male embryos vs. 86.9±8.1 hours in female embryos, p = 0.06) and tB (100.2±5.9 hours versus 97.9±6.5 hours, p = 0.07). Male embryos showed an increased average time of 2.7 hours to tSB and 2.3 hours to tB, while no differences were found in the mean times of all the other morphokinetic paraments measured (p &amp;gt; 0.50): tPNf (∼21.8±3.0 hours) t2 (∼24.4±3.2 hours); t3 (∼35.6±3.9 hours); t4 (∼36.6±4.6 hours); t5 (∼46.9±6.0 hours); t6 (∼53.5±7.0 hours); t7 (∼54.1±7.3 hours) and t8 (∼54.1±7.3 hours). This finding suggests a sex-specific difference in reaching blastocyst stages. Limitations, reasons for caution The main limitation of the study is its retrospective nature and the small sample size. We analyzed the data of embryos leading to a live birth (high-quality embryos), therefore, caution should be made when generalizing results to non-implanting embryos (of potentially lower quality). Wider implications of the findings: Sex-specific differences in developmental timings of preimplantation embryos at blastocyst stage, as evidenced by time-lapse data, should be considered to avoid selection biases during embryo transfers in ART clinic. Trial registration number Not applicable

Round Spermatid Injection Rescues Female Lethality of a Paternally Inherited Xist Deletion in Mouse.

In mouse female preimplantation embryos, the paternal X chromosome (Xp) is silenced by imprinted X chromosome inactivation (iXCI). This requires production of the noncoding Xist RNA in cis, from the Xp. The Xist locus on the maternally inherited X chromosome (Xm) is refractory to activation due to the presence of an imprint. Paternal inheritance of an Xist deletion (XpΔXist) is embryonic lethal to female embryos, due to iXCI abolishment. Here, we circumvented the histone-to-protamine and protamine-to-histone transitions of the paternal genome, by fertilization of oocytes via injection of round spermatids (ROSI). This did not affect initiation of XCI in wild type female embryos. Surprisingly, ROSI using ΔXist round spermatids allowed survival of female embryos. This was accompanied by activation of the intact maternal Xist gene, initiated with delayed kinetics, around the morula stage, resulting in Xm silencing. Maternal Xist gene activation was not observed in ROSI-derived males. In addition, no Xist expression was detected in male and female morulas that developed from oocytes fertilized with mature ΔXist sperm. Finally, the expression of the X-encoded XCI-activator RNF12 was enhanced in both male (wild type) and female (wild type as well as XpΔXist) ROSI derived embryos, compared to in vivo fertilized embryos. Thus, high RNF12 levels may contribute to the specific activation of maternal Xist in XpΔXist female ROSI embryos, but upregulation of additional Xp derived factors and/or the specific epigenetic constitution of the round spermatid-derived Xp are expected to be more critical. These results illustrate the profound impact of a dysregulated paternal epigenome on embryo development, and we propose that mouse ROSI can be used as a model to study the effects of intergenerational inheritance of epigenetic marks.

Regulation of X-linked gene expression during early mouse development by Rlim.

Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos.

Male and female embryos differ in their response to oxygen concentration

Male and female preimplantation embryos exhibit differences in their physiologies due to the presence of a second active X-chromosome in female embryos prior to implantation. Here we evaluate the impact of oxygen concentration on the morpho-kinetic development of male and female mouse embryos. Time-lapse analysis of embryo development, followed by determination of embryo sex and blastocyst cell number after embryo culture in either 5% or 20% oxygen. Male C57BL/6 mice with an X-linked enhanced green fluorescent protein (eGFP) were used as studs to create females that expressed eGFP from the 8-cell embryo stage. Pronucleate oocytes (n=500) were cultured individually for 48 h in 2 μL drops of G1, followed by 48h in G2, under paraffin oil at 37°C in a multi-gas imaging incubator (Sanyo MCOK-5M[RC]) set to 6% CO2, 5% O2, 89% N2 or 6% CO2 in air (20% oxygen). Morphology of each embryo was assessed every 15 minutes and resultant blastocyst cell number determined. In 5% oxygen the times between divisions of male embryos were similar to females. However, in 20% oxygen female embryo development was characterized by significant delays in cleavage times from the 7-cell stage onwards (P< 0.05), culminating in delayed time of blastocoel formation (P<0.01). Mean blastocyst cell number for male and female embryos in 5% oxygen were 113.6 ± 2.1 and 107.5 ± 2.1 respectively (P<0.05; a difference of 3.6%). At 20% oxygen, blastocyst cell numbers for male and female embryos were 40.3 ± 2.0 and 34.7 ± 2.1 respectively (P<0.01; a difference of 14.0%). Confirming previous reports, it was found that atmospheric oxygen had an adverse effect on the developmental kinetics of the preimplantation mouse embryo. It was further determined that development of female embryos not only lagged behind their male counterparts, but that female embryos appear to be more sensitive to a high oxygen concentration.

Sex-specific embryonic origin of postnatal phenotypic variability

Preimplantation developmental plasticity has evolved in order to offer the best chances of survival under changing environments. Conversely, environmental conditions experienced in early life can dramatically influence neonatal and adult biology, which may result in detrimental long-term effects. Several studies have shown that small size at birth, which is associated with a greater risk of metabolic syndrome, is largely determined before the formation of the blastocysts because 70%-80% of variation in bodyweight at birth has neither a genetic nor environmental component. In addition, it has been reported that adult bodyweight is programmed by energy-dependent process during the pronuclear stage in the mouse. Although the early embryo has a high developmental plasticity and adapts and survives to adverse environmental conditions, this adaptation may have adverse consequences and there is strong evidence that in vitro culture can be a risk factor for abnormal fetal outcomes in animals systems, with growing data suggesting that a similar link may be apparent for humans. In this context, male and female preimplantation embryos display sex-specific transcriptional and epigenetic regulation, which, in the case of bovine blastocysts, expands to one-third of the transcripts detected through microarray analysis. This sex-specific bias may convert the otherwise buffered stochastic variability in developmental networks in a sex-determined response to the environmental hazard. It has been widely reported that environment can affect preimplantation development in a sex-specific manner, resulting in either a short-term sex ratio adjustment or in long-term sex-specific effects on adult health. The present article reviews current knowledge about the natural phenotypic variation caused by epigenetic mechanisms and the mechanisms modulating sex-specific changes in phenotype during early embryo development resulting in sex ratio adjustments or detrimental sex-specific consequences for adult health. Understanding the natural embryo sexual dimorphism for programming trajectories will help understand the early mechanisms of response to environmental insults.

X-inactivation and X-reactivation: epigenetic hallmarks of mammalian reproduction and pluripotent stem cells.

X-chromosome inactivation is an epigenetic hallmark of mammalian development. Chromosome-wide regulation of the X-chromosome is essential in embryonic and germ cell development. In the male germline, the X-chromosome goes through meiotic sex chromosome inactivation, and the chromosome-wide silencing is maintained from meiosis into spermatids before the transmission to female embryos. In early female mouse embryos, X-inactivation is imprinted to occur on the paternal X-chromosome, representing the epigenetic programs acquired in both parental germlines. Recent advances revealed that the inactive X-chromosome in both females and males can be dissected into two elements: repeat elements versus unique coding genes. The inactive paternal X in female preimplantation embryos is reactivated in the inner cell mass of blastocysts in order to subsequently allow the random form of X-inactivation in the female embryo, by which both Xs have an equal chance of being inactivated. X-chromosome reactivation is regulated by pluripotency factors and also occurs in early female germ cells and in pluripotent stem cells, where X-reactivation is a stringent marker of naive ground state pluripotency. Here we summarize recent progress in the study of X-inactivation and X-reactivation during mammalian reproduction and development as well as in pluripotent stem cells.

Sex determines the expression level of one third of the actively expressed genes in bovine blastocysts

Although genetically identical for autosomal Chrs (Chr), male and female preimplantation embryos could display sex-specific transcriptional regulation. To illustrate sex-specific differences at the mRNA level, we compared gene-expression patterns between male and female blastocysts by DNA microarray comparison of nine groups of 60 bovine in vitro-produced blastocysts of each sex. Almost one-third of the transcripts detected showed sexual dimorphism (2,921 transcripts; false-discovery rate, P < 0.05), suggesting that in the absence of hormonal influences, the sex Chrs impose an extensive transcriptional regulation upon autosomal genes. Six genes were analyzed by qPCR in in vivo-derived embryos, which displayed similar sexual dimorphism. Ontology analysis suggested a higher global transcriptional level in females and a more active protein metabolism in males. A gene homolog to an X-linked gene involved in network interactions during spliceosome assembly was found in the Y-Chr. Most of the X-linked-expressed transcripts (88.5%) were up-regulated in females, but most of them (70%) exhibited fold-changes lower than 1.6, suggesting that X-Chr inactivation is partially achieved at the blastocyst stage. Almost half of the transcripts up-regulated in female embryos exhibiting more than 1.6-fold change were present in the X-Chr and eight of them were selected to determine a putative paternal imprinting by gene expression comparison with parthenogenetic embryos. Five (BEX, CAPN6, BEX2, SRPX2, and UBE2A) exhibited a higher expression in females than in parthenotes, suggesting that they are predominantly expressed by the paternal inherited X-Chr and that imprinting may increase the transcriptional skew caused by double X-Chr dosage.

X Chromosome Inactivation Is Initiated in Human Preimplantation Embryos

X chromosome inactivation (XCI) is the mammalian mechanism that compensates for the difference in gene dosage between XX females and XY males. Genetic and epigenetic regulatory mechanisms induce transcriptional silencing of one X chromosome in female cells. In mouse embryos, XCI is initiated at the preimplantation stage following early whole-genome activation. It is widely thought that human embryos do not employ XCI prior to implantation. Here, we show that female preimplantation embryos have a progressive accumulation of XIST RNA on one of the two X chromosomes, starting around the 8-cell stage. XIST RNA accumulates at the morula and blastocyst stages and is associated with transcriptional silencing of the XIST-coated chromosomal region. These findings indicate that XCI is initiated in female human preimplantation-stage embryos and suggest that preimplantation dosage compensation is evolutionarily conserved in placental mammals.

Analysis of the sex ratio in preimplantation embryos from B6.K1 and B6.K2 Ped gene congenic mice

The mouse preimplantation embryo development (Ped) gene product, Qa-2, which is the homolog of human HLA-G, influences the rate of preimplantation embryonic development and overall reproductive success. The sex ratio in preimplantation embryos from Ped gene congenic mice was examined in order to determine whether embryo sex is a confounding factor in the control of the rate of preimplantation development. B6.K1 (Ped slow) and B6.K2 (Ped fast) congenic mice differ only in the absence (B6.K1) or presence (B6.K2) of the genes encoding Qa-2 protein. We analyzed the sex of B6.K1 (n=221) and B6.K2 (n=260) preimplantation embryos by using Real-Time PCR with primers specific for the X and Y chromosomes. We found that there was no statistically significant difference in the ratio of male to female preimplantation embryos in either strain. We conclude that the sex of the embryos is not a confounding factor that affects the Ped gene control of the rate of preimplantation development. Therefore, the Ped gene is entirely responsible for mediating the faster development of B6.K2 embryos compared to B6.K1 embryos.

Differential sensitivity of male and female mouse embryos to oxidative induced heat-stress is mediated by glucose-6-phosphate dehydrogenase gene expression

During the preimplantation period, in vitro cultured males have a higher metabolic rate, different gene expression, and grow faster than females. It has been suggested that under some stress conditions male embryos are more vulnerable than females; however, the biological fragility of male embryos is little understood. Since many forms of stress result in the overproduction of cellular reactive oxygen species (ROS), we addressed the hypothesis that the connection between female advantage during early developmental stages and heat stress involves ROS and differential gene expression of G6PD, an X-linked gene related to oxidative stress. We have found that after compaction, female heat-stressed embryos have less relative amounts of H2O2 than males, and female embryos survive better than males under in vivo or in vitro heat stress situations. In addition, in vitro produced female embryos grow slower than male embryos, have differential mRNA transcription of G6PD and also of some genes situated on autosomal-chromosomes (Sox, Bax, and Oct-4). Moreover, by inhibiting G6PD, all differences generated by oxidative stress between male and female embryos disappear. For the first time, we provide an experimental demonstration of a mechanism that explains why following exposure to heat stress-induced ROS, female preimplantation embryos are more resistant than males.

Expression of an Xist promoter-luciferase construct during spermatogenesis and in preimplantation embryos: regulation by DNA methylation.

Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of the two X chromosomes in females, a process involving a regulatory gene, Xist (X-inactive specific transcript). Xist maps to the X-inactivation centre and is expressed from the inactive X chromosome in female somatic cells and at the time of X inactivation during spermatogenesis in the male. In female preimplantation embryos, Xist demonstrates imprinting in that the paternal allele inherited from the sperm is preferentially expressed. This preferential paternal Xist expression is correlated with paternal X inactivation in the extraembryonic lineages at the blastocyst stage. We have analysed a 233-bp Xist promoter fragment (nt -220 to +13) for its ability to direct appropriate expression and its regulation by DNA methylation. This minimal promoter sequence directs expression of the luciferase reporter gene following injection of the construct into one-cell embryos. In vitro methylation of the construct before injection represses transcription. In six different transgenic lines, expression of the Xist promoter-luciferase transgene occurs only in the testis of the males (as for the endogenous Xist gene). The testis-specific expression is correlated with hypomethylation of the transgene, although to different extents in different lines. Following paternal transmission, expression of the Xist promoter-luciferase construct in preimplantation embryos is correlated with degree of hypomethylation in the testis and the degree of hypomethylation of the transgene in embryos at the morula stage. It is concluded that the patterns of methylation of the transgene in sperm (and in microinjected transgenes) can regulate the activity of the Xist promoter in the preimplantation embryo and thus support the hypothesis that gametic methylation patterns govern imprinted expression of the endogenous Xist gene in development.

Timing of Mating, Developmental Asynchrony and the Sex Ratio in Mice

Krackow, S. and P. S. Burgoyne. Timing of mating, developmental asynchrony and the sex ratio in mice. PHYSIOL BEHAV 63(1): 81–84, 1998.—According to the developmental asynchrony hypothesis, changing the time of mating within the estrous cycle could alter the interval between completion of blastocyst development and uterine responsiveness for implantation. This may then lead to sex ratio skews in animals that exhibit sex-differential blastocyst development, because uterine stage may now benefit either slow (female) or fast (male) developing blastocysts. To test this hypothesis, the responses of two strains of mice to altered mating dynamics were compared. In a strain that exhibits higher male than female blastocyst developmental rates, sex ratios became significantly female-biased when mated late during the estrous cycle as opposed to early mating. However, timing of mating did not affect sex ratios in a strain with synchronous development of male and female preimplantation embryos. Hence, it is concluded that developmental asynchrony between male and female blastocysts on the one hand, and blastocysts and uterus on the other, are indeed responsible for the effect of timing of mating on litter sex ratios in mice.

Does sex determination start at conception?

Recent molecular studies of mammalian sexual determination have been focused on gene expression in the gonadal ridge at the time of appearance of sexual dimorphism: the critical time defined by the 'Jost principle'. Three lines of evidence suggest that, instead, sex determination may start shortly after conception: (1) the XY preimplantation embryo usually develops more rapidly than the XX preimplantation embryo (this phenotype has been linked to the Y chromosome and will be termed 'Growth factor Y'); (2) the gene for testis determination, SRY/Sry, and the closely linked genes ZFY/Zfy and Smcy, are transcribed in the preimplantation embryo; and (3) male and female preimplantation embryos are antigenically distinguishable, indicating sex differences in gene expression. The data to support these assertions are reviewed. Possible relationships of these three phenomena to each other and to sex differentiation are discussed. Similarities in mechanisms of sexual determination between marsupial and eutherian mammals are hypothesized. Problems with interpreting male sexual differentiation as being solely due to testosterone and Müllerian inhibiting substance (MIS) are discussed.

XIST Expression in Human Oocytes and Preimplantation Embryos

During mouse preimplantation development, the exclusive expression of the Xist gene from the paternally inherited allele is thought to play a role in the inactivation of the paternally inherited X chromosome in the extra-embryonic cell lineages of the developing female embryo. Recently, inactivation of the paternally inherited X chromosome has also been shown to occur in the extraembryonic cell lineages of the human female conceptus. In this paper, we determine whether the pattern of XIST expression in human preimplantation embryos is similarly correlated with paternal X inactivation. We developed procedures sensitive to the single cell, for the simultaneous analysis of XIST and HPRT expression and of sexing, initially using human fibroblast cells. Application of these procedures to human cleavage-stage embryos derived by in vitro fertilization revealed a pattern of XIST expression different from that in the mouse. Transcripts of the XIST gene were detected as early as the 1-cell zygote and, with increasing efficiency, through to the 8-cell stage of preimplantation development. In addition, transcripts of XIST were detected in both male (hence from the maternally inherited allele) and female preimplantation embryos. This pattern of expression is not consistent with a role for the early expression of the XIST gene in the choice of paternal X inactivation in the extraembryonic cell lineages of the developing human embryo.

Preimplantation sexing and diagnosis of hypoxanthine phosphoribosyl transferase deficiency in mice by biochemical microassay

Hypoxanthine phosphoribosyl transferase (HPRT)-deficient male embryos derived from heterozygous (carrier) female mice were diagnosed by biochemical microassay of X-chromosome-coded HPRT activity in a single cell taken from the 8-cell embryo or in 5-10 cells sampled from the blastocyst. In the latter procedure, carrier female blastocysts could also be distinguished from affected males, and normal males and females, as having intermediate HPRT activity in the sampled trophectoderm cells. During the assay procedures, the operated preimplantation embryos were cultured. They were then transferred, in batches as diagnosed, to recipient females. The resulting fetuses were sexed by gonad morphology and assayed for HPRT activity. All those identified as HPRT-negative embryos by biopsy at the 8-cell or blastocyst stages were indeed HPRT-negative males. The heterozygous females were also correctly identified by the trophectoderm biopsy procedure. The sex of an embryo can also be diagnosed by HPRT activity dosage in a single blastomere taken from 8-cell embryos from a normal mating and cultured for 12 hours before assay. Both X chromosomes are active in female morulae and the blastomeres sampled from female preimplantation embryos have twice the X-coded HPRT activity compared to those from the male embryos. The accuracy of this procedure for sexing was again verified by transfer of the putative male and putative female embryos into recipient females.