Blastocyst Fluid Research Articles

A prospective study to evaluate the gender prediction of blastocysts by using cell-free DNA within a culture medium.

BackgroundPreimplantation genetic diagnosis (PGD) has been used as an option for couples with the possibility of having a baby with a genetic disorder. The common method for performing this test involves isolating 1 cell from day 3 or a few cells from day 5 embryos and performing genetic studies on the cell-extracted DNA. This method is invasive and can cause abortion after implantation in the uterus. Because of this, 2 noninvasive methods for performing a PGD have been studied: PGD using blastocyst fluid and PGD using embryo culture medium.ObjectiveThe aim of this study is to determine the sensitivity of the polymerase chain reaction (PCR) technique to detect the Y chromosome using cell-free DNA within a culture medium for gender prediction of blastocysts.Materials and Methods In this study, the gender of 30 embryos on day 5 was determined using embryonic DNA extraction from the culture medium and the PCR technique to evaluate the sex-determining region Y and fragile X mental retardation genes. Then, the accuracy was assessed using ultrasound.ResultsThe results of the PCR technique showed that 7 embryos were male, but an ultrasound revealed that 13 were male.ConclusionThe given results indicated that, because of the low amount of DNA extracted from the culture medium, the diagnosis of the existence of the Y chromosome by this method is still not accurate enough for detecting the gender of the embryo.

Non-invasive chromosome screening for embryo preimplantation using cell-free DNA

Abstract Preimplantation genetic testing (PGT) is a widely adopted screening method that can be performed to identify and select embryos with normal ploidy; however, PGT relies on embryo biopsy, that is, polar body, embryo cells, or trophectoderm biopsy, to obtain embryonic DNA, increase its technical limitations. Studies have indicated that biopsy may have an influence on the quality and development of embryos, and increase the chance of abnormal epigenetic modifications. Therefore, non-invasive PGT (niPGT) detection of cell-free DNA (cfDNA) has gradually become a hot research topic in the field of assisted reproduction. Studies showed cfDNA could be detected in blastocyst fluid and spent culture medium (SCM) in vitro cultured embryos. The cfDNA collection requires less skill and makes lower risk to embryos. Some studies have been conducted to evaluate the feasibility of SCM-based niPGT approaches. When comparing the ploidy consistency of cfDNA in SCM, its consistency to the conventional PGT for aneuploidies results fluctuated widely, it is critical to recognize the factors influencing accuracy. These contradictory results may be related to factors such as the difference in SCM sampling methods and sampling time, and the definition of consistency. In this review, we aimed to comprehensively summarize how researchers use embryonic cfDNA to conduct niPGT detection. It also systematically reviews the factors affecting the accuracy of the test and its underlying issues, as well as prospective applications. We hope to provide a basis for future niPGT research and a useful reference for the standardized operation of niPGT that can be widely applied in clinical practice.

P–516 Evidence for self-correction in preimplantation embryos by comparative molecular karyotyping of blastocoele fluid, trophectoderm and inner cell mass

Abstract Study question Does the molecular karyotype of the cell-free DNA (cfDNA) from the blastocyst fluid (BF) can predict the efficiency of self-correction of karyotype of preimplantation embryo? Summary answer Detection of aneuploidies in the BF potentially can point out on effective self-correction of blastocyst karyotype and consequently on high developmental potential of mosaic embryos. What is known already Correction of aneuploidies in the preimplantation embryos can be provided by several mechanisms, including apoptosis. The predominant death of aneuploid cells was demonstrated in mouse embryos (Bolton, 2016). A positive correlation was also shown between the concentration of cfDNA from the BF of human blastocyst and the morphology of the embryo, as well as between the activity of caspase–3 and the concentration of cfDNA (Rule, 2018). The incidence of failed amplification after WGA being significantly higher among euploid blastocysts (Magli, 2019). The capacity of abnormal cells extruding into the BF would be related to the embryo development potential (Gianaroli, 2019). Study design, size, duration This is a prospective observational study of thirty-one Day 5 human blastocysts. Cryopreserved blastocysts were received after treatment cycles at the IVF Center with informed consent obtained from couples. The average age of 15 women was 32.25±5 years. The morphological characteristics of blastocysts were estimated in accordance with the Gardner classification (Gardner, Schoolcraft, 1999). The procedure of BF aspiration and trophectoderm (TE) and ICM cells separation of the blastocysts was previously described (Tsuiko, 2018). Participants/materials, setting, methods WGA was performed by PicoPLEX kit (Rubicon Genomics, USA) or REPLI-g Mini kit (Qiagen) according to manufacturer’s protocols. The DNA of the BF, ICM and TE were analyzed separately using cCGH, aCGH and NGS. SurePrint G3 Human CGH Microarrays (8x60K, Agilent Technologies) were used according to the manufacturer’s recommendations. Image analysis was done using ISIS (v.5.5) (Metasystems) and Agilent CytoGenomics Software (v.3). VeriSeq™ PGS Kit - MiSeq® System (Illumina) was used for NGS. Main results and the role of chance Molecular karyotypes of all three samples - BF, ICM and TE, were obtained for 23 (74.2%) blastocysts. A correlation between the woman’s age and the number of aneuploidies in cfDNA (p = 0.0009) was found. A positive correlation may indicate that the number of aneuploidies in the embryonic cells increases with the age of a woman, however, the embryonic karyotype undergoes self-correcting through the elimination of aneuploid cells. It was noted that well-developing blastocysts (groups 4–5, according to Gardner’s classification) had fewer aneuploidies in ICM (p = 0.0141) and TE (p = 0.0436). In contrast, there was a tendency to an increase in the number of aneuploidies in the BF during blastocysts transition from stage 3 to 5 (p = 0.3542). We assessed the relationship between the number of aneuploidies in groups of blastocysts with different characteristics of ICM (groups “A” and “B” according to Gardner’s classification). These groups significantly differ in the number of aneuploidies in cfDNA (p = 0.0352), although the statistically significant differences between the number of aneuploidies in ICM (p = 0.5992) and in TE (p = 0.5934) was not detected. Thus, higher-quality embryos in terms of ICM morphology contain more abnormalities in the BF, since in this group the elimination of aneuploid cells is more efficient. Limitations, reasons for caution The number of embryos is limited in this study. More comprehensive studies are required to confirm the observed tendency. Wider implications of the findings: Aneuploid cells elimination can be a cause of increasing cfDNA concentration in the BF, which may be a marker of the viability of mosaic embryos when it is necessary to decide on mosaic embryo transfer. This study was supported by the RFBR (15–04–08265) and by the RSF (20–74–00064). Trial registration number Not applicable

Relationship between blastocoel cell-free DNA and day-5 blastocyst morphology.

Cell-free DNA (cfDNA) which is present in the blastocoel cavity of embryos is believed to result from physiological apoptosis during development. This study assessed cfDNA content and caspase-3 protease activity in day-5 IVF blastocysts to determine if there was a correlation with embryo morphology. Day-5 IVF blastocysts were scored according to the Gardner and Schoolcraft system (modified to generate a numerical value) and cfDNA was collected following laser-induced blastocoel collapsing prior to cryopreservation in 25μL of media. cfDNA was quantified via fluorospectrometry and apoptotic activity was assessed via a caspase-3 protease assay using a fluorescent peptide substrate. Data were compared by linear regression. A total of 32 embryos were evaluated. There was a significant (p < 0.01) and positive correlation (cfDNA = 104.753 + (11.281 × score); R2 = 0.200) between embryo score and cfDNA content. A significant (p < 0.05) and positive correlation (cfDNA = 115.9 + (0.05 × caspase-3); R2= 0.128) was observed between caspase-3 activity and cfDNA levels. There was no significant relationship between caspase-3 activity and embryo morphology score. This study provides further evidence that cfDNA is present in blastocoel fluid, can be quantified, and positively correlates with embryonic morphology. There is also evidence that at least a portion of the cfDNA present is from intracellular contents of embryonic cells that underwent apoptosis. Additional studies are warranted to determine other physiological sources of the cfDNA in blastocyst fluid and to determine the relationship with cfDNA content, embryo morphology, and chromosomal ploidy status plus implantation potential.

Evidence for the presence of sodium- and potassium-dependent adenosine triphosphatase alpha1 and beta1 subunit isoforms and their probable role in blastocyst expansion in the preattachment horse conceptus.

The unusual hypotonicity of equine blastocyst fluid has prompted us to investigate the role of sodium- and potassium-dependent adenosine triphosphatase (Na+,K+-ATPase) in the process of fluid accumulation in the horse conceptus. Nine mares were used for the experiments. Reverse transcriptase polymerase chain reaction was conducted on two sets of five conceptuses recovered between 12 and 28 days (+/- 1 day) after ovulation. Messenger RNAs encoding the alpha1 and beta1 subunit isoforms of Na+,K+-ATPase were detected in all embryonic tissues examined. Western blot analysis showed that alpha1 and beta1 subunits are both present in Day 15 conceptuses. Trophoblast tissues from 19 conceptuses between 8 and 31 days after ovulation were stained immunohistochemically using primary antibodies against the alpha1 and beta1 subunit isoforms of the Na+,K+-ATPase. Both isoforms were detected in all sections. Trophoblastic vesicles, prepared from 6 conceptuses between 12 and 14 days after ovulation, were used to investigate the inhibition of blastocyst expansion with ouabain after collapse induced with cytochalasin D. In normal medium there was a mean 3-fold increase, and in ouabain (10(-6) M) a mean 3-fold decrease, in the volume of vesicles that had been partially collapsed with cytochalasin D. We therefore conclude that, despite the hypotonicity of the blastocyst fluid in the early horse conceptus, the Na+,K+-ATPase plays a role in its accumulation, as in other species.

Osmolality of equine blastocyst fluid from day 11 to day 25 of pregnancy.

Horse conceptuses collected between Day 11 and Day 18 of pregnancy float in isotonic media. To investigate this phenomenon, blastocyst fluids from 30 conceptuses from 13 mares were analysed for osmolality and for concentrations of Na+, Cl-, K+, glucose, urea and creatinine. In conceptuses from Group A, samples from Day 11 to Day 16 yielded the following results (mean +/- s.e.m.): osmolality, 121.4 +/- 1.5 mOsm kg-1; Na+, 11.0 +/- 2.2 mM; Cl-, 29.3 +/- 2.5 mM; K+, 26.2 +/- 2.6 mM; glucose, 0.6 +/- 0.1 mM; urea, 6.0 +/- 0.6 mM; creatinine, 9.6 +/- 1.1 microM. Between Day 16 and Day 25, the osmolalities and Na+ concentrations increased gradually with age but the former never exceeded 255 mOsm kg-1. Fluids from Group B were obtained from eight conceptuses exposed to saline solutions of different osmolalities for various periods of time. An increased perivitelline space of 1-3 mm became evident at the lower pole of the floating conceptus after 45 min of exposure to solutions with osmolalities of > or = 300 mOsm kg-1, suggesting that Na+ and Cl- diffuse freely through the capsule but not through the trophoblast. The significance of the hypo-osmolality of equine blastocyst fluid is discussed but remains unclear.

Changes in the osmolality of equine blastocyst fluid between days 11 and 25 of pregnancy

With the increase in the penetration of renewable energy resources and advances in metering, communication and edge computing infrastructure, the importance and value of customer-side resources for the provision of transmission-level and distribution-level grid services has increased significantly. As such, this article provides a thorough review on different proposed modeling and control methodologies for selected customer-side resources and examines how each methodology meets the requirement for certain grid services both at the transmission and distribution levels. The services are considered from a competitive markets perspective. Specifically, commercial and residential heating, ventilation and air-conditioning systems (HVACs), water heating units, refrigeration units, batteries and electric vehicles (EVs) are the resources considered. Services considered include frequency regulation, spinning reserves provision, voltage management, deferral of transmission and distribution grid capacity upgrades, reliability and resiliency improvement. Finally, discussions on research needs regarding the usage of groups of heterogeneous resources for cascaded services are presented.

Role of chloride transport in the development of the rat blastocyst.

Preimplantation mammalian development culminates in the generation of a fluid-filled cavity, the blastocoel, which requires the vectorial transport of ions across the trophectoderm, followed by the movement of water. Experiments were carried out to establish the role of Cl- transport in blastocoel formation in the rat. These included investigations of the effect of Cl- substitution and the Cl- transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, furosemide, and anthracene-9-carboxylic acid, on the development of morulae into blastocysts in culture, and on the rate at which blastocoel fluid is accumulated. In addition, a novel technique was developed in which the Cl(-)-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl) quinolium (SPQ) was loaded into the blastocoel cavity, in order to characterize the pathways of transtrophectodermal Cl- transport. We established that 1) blastocoel formation in the rat requires the external presence of Cl- ions; 2) transport across the trophectoderm appears to be via a transcellular pathway, since rates of blastocyst development, fluid accumulation, and SPQ-monitored Cl- efflux are all reduced in the presence of Cl- transport inhibitors; and 3) Cl- transport occurs via Cl-/HCO3- exchange.

Variations of lectin-binding glycoproteins in early pregnant rabbit embryo.

In the present study, the quantitative and qualitative changes of three kinds of lectin-binding glycoproteins of early pregnant rabbit embryos (D4-D12) were analyzed. The technique of Western blot, as well as video densitometer scanning and its analytic software was used for analysis. Results found that there were five specific lectin-binding glycoproteins in Day 4 to Day 6 blastocyst fluids: one ConA-binding glycoprotein about 70 kD, two WGA-binding glycoproteins respectively about 42 kD and 25 kD and two PNA-binding glycoprotein respectively about 180 kD and 75 kD. They disappeared immediately after implantation. It is demonstrated that there are stage-specific glycoproteins in rabbit blastocyst fluid which might be relevant to the recognition of pregnancy and implantation.

Uteroglobin in the developing rabbit conceptus in vivo and in vitro

Uteroglobin (UGL) was measured in day-4 to day-10 rabbit conceptuses by a competitive ELISA. Levels in blastocyst fluid, tissues, coverings and in the early fetus were determined separately. The total amount of UGL increased from 18.4 ng to 6.8 micrograms per conceptus. The UGL content of individual day-6 blastocysts was studied in vitro. Culturing was carried out up to 60 h in Ham's F10 medium with polyvinylpyrrolidone as macromolecular component, with and without progesterone, and with progesterone plus estradiol. UGL was determined in the blastocyst fluids, tissues with coverings and in the culture media. After labelling with [35S]-methionine, protein patterns of total blastocysts and of culture media were analysed by two-dimensional gel electrophoresis and fluorography. The morphology of cultured blastocysts was examined by electron microscopy. During 60 h of culture, the blastocysts expanded in diameter by 84%, and released 19% of their initial UGL content into the medium, independent of the hormonal substitution. Neither de novo synthesis, nor degradation of UGL was found: the protein remained unlabelled in fluorography, and its total quantity was not significantly different from that of non-cultured controls. Trophoblast, endoderm and embryoblast cells showed well preserved cell organelles and intercellular junctions, while the morphological differentiation of the germ layer was inhibited.

Ultrastructure, protein synthesis and secretion of day-6 rabbit blastocysts cultured in a chemically defined, protein-free medium

Day-6 rabbit blastocysts were cultured in Ham's F10 medium supplemented with polyvinylpyrrolidone as a macromolecular component, for 4 to 12 h. The integrity of the blastocyst cells was demonstrated by electron microscopy. Expansion and biosynthesis of proteins and of DNA were studied after culturing in the presence of 35S-methionine and 3H-thymidine. Polyvinylpyrrolidone did not interfere with the subsequent protein analysis, which was performed by two dimensional gel electrophoresis followed by silver staining and fluorography. More than 600 labelled proteins were found in the blastocyst tissue, many of them were also present in the blastocyst fluid and in the blastocyst coverings. Several proteins seemed to be produced for incorporation into the blastocyst coverings; others, only detected in the culture medium, might have been synthesized for secretion into the environment.

Utilization and absorption of carbohydrates by the plerocercus metacestode of otobothrium insigne (cestoda: Trypanorhyncha)

Hildreth M. B. and Lumsden R. D. 1988. Utilization and absorption of carbohydrates by the plerocercus metacestode of Otobothrium insigne (Cestoda:Trypanorhyncha). International Journal for Parasitology 18: 251–257. Otobothrium insigne plerocerci were used to study the nutritional relationship between a metacestode bladder and its juvenile scolex relative to carbohydrate metabolism. This plerocercus contains a large quantity of glycogen in its blastocyst (bladder) outer wall and juvenile scolex. Blastocyst fluid contains a large amount of glucose, at a concentration significantly higher than that of the host's serum. When scoleces were excised from their blastocysts and maintained in saline without glucose, their glycogen content decreased significantly. However, if the scoleces were left in their blastocysts during maintenance in saline, their glycogen reserve remained unchanged, and the glycogen reserve in the blastocyst decreased significantly. This suggests that the blastocyst may function as a glucose reservoir for the scolex. The blastocyst absorbs glucose exclusively by active transport; in contrast, glucose is absorbed into the scolex by active transport and diffusion. The elevated glucose concentration in the blastocyst may cause glucose to enter the scolex primarily by diffusion. Therefore, the blastocyst may also serve to push glucose into the scolex by creating a high diffusion gradient for glucose in the fluid just external to the scolex.

Partial purification and characterization of a trypsin-like proteinase from rabbit preimplantation uterine fluid.

Previous investigations have proved that proteinases are involved in implantation of the rabbit embryo into the uterine tissues. This study describes a trypsin-like enzyme found in the blastocyst fluid and uterine flushings at the time of implantation. The proteinase isolated from uterine flushings has a molecular mass of about 50 kDa and exists in two differently charged forms of pI 4.0 and 4.5. Tests with low molecular mass 4-nitroanilide substrates proved a marked cleavage selectivity of the enzyme for arginyl bonds. The catalytic activity is not affected by Ca2+ and EDTA but inhibited by aprotinin and a high concentration (10(-6)M) of lima bean trypsin inhibitor.

Chorionic gonadotropin receptors and immunoreactive chorionic gonadotropin in implantation of the rabbit blastocyst

To determine the temporal relationship between immunoreactive chorionic gonadotropin, chorionic gonadotropin receptors, and implantation of the rabbit blastocyst, (1) immunoreactive chorionic gonadotropin in plasma, uterine flushings and blastocysts; (2) chorionic gonadotropin receptors on blastocysts (day 5 and 6) and embryonic and interembryonic segments of the uterus (day 7); and (3) chorionic gonadotropin receptors on the endometrium and myometrium (day 1 through 6) were measured. Binding of I125-labeled β-subunit of human chorionic gonadotropin (hCG) by cell membranes from blastocysts increased significantly from 6.0 ± 1.1 fmol/mg of protein (mean ± SE) on day 5 (N = 6) to 16.1 ± 1.3 fmol/mg of protein on day 6 (n = 6) (P < 0.001). Immunoreactive chorionic gonadotropin levels in blastocyst fluid increased from 0.3 ng/ml of day 5 to 0.65 ng/ml on day 6. Specific binding of I125-labeled hCG was absent in endometrial and myometrial cell membranes before implantation (days 1 to 6) but was found in decidual cells from embryonic segments on day 7. Plasma immunoreactive chorionic gonadotropin or chorionic gonadotropin-like material increased from 6 ng/ml of day 1 to 52 ng/ml on day 7. Uterine flushings had chorionic gonadotropin levels of 0.4 ng/ml of day 2, which increased slowly to 1.0 ng/ml on day 7. Intrauterine instillation of hCG into nonpregnant uterine horns demonstrated transuterine absorption of hCG with plasma hCG levels showing a dose-related response. Our findings demonstrate that (1) immunoreactive chorionic gonadotropin or chorionic gonadotropin-like material is detectable in plasma, uterine flushings, and blastocyst fluid before implantation, (2) chorionic gonadotropin receptors are present on the blastocyst cells before implantation, and (3) the uterus can absorb chorionic gonadotropin from its lumen.

Effects of copper and zinc on rat uterine muscle contraction and rabbit blastocyst fluid accumulation.

The effect of copper and zinc on the isometric contractility of isolated rat uterine muscle has been studied. Results show that concentrations of 2 X 10 #{176}M Cuproduce a drastic potentiating effect on the contraction, elicit spontaneous contractile activity, and at concentrations higher than 8 X 10 #{176}M, produce spasmodic contracture of the muscle. Effects of Cuare reversible. Zinc, at concentrations of 2 to 4 X 10- #{176}M has a marked depressant effect on rat uterine contractility. Although the effect of Znis not reversed by washing under our experimental conditions, Cuin equivalent concentrations can reverse the depressant effect of Zn�. The potentiating effect of Cu� is not modified by Zn�#{176} at equimolar concentrations. Rabbit blastocyst fluid accumulation is not altered by 2 to 5 X 10 5M Cuor Znconcentrations. However, long-term incubation (>1 h) in solutions containing 5 X 10 #{176}M Curesults in complete degeneration of the embryo. The relevance of these results in relation to decreased expulsion rate and increased contracep- tive effect of CuTIIJD is discussed.

Gonadotropin-Like Substance in the Preimplanted Rabbit Blastocyst

The presence of a gonadotropin-like substance in preimplanted rabbit blastocyst fluid was determined bu radioreceptorassay of human chorionic gonadotropin (hCG)/luteinizing hormone (LH), using receptors prepared from bovine corpus luteum, rat testis, and rat ovaries. An average of 16.6 microliter of fluid containing 0.83 ng of luteotropic material was recovered from each blastocyst. An intense fluorescence was exhibited by the trophoblastic cells of the blastocysts treated with fluorescein-conjugated gamma-globulin isolated from antiserum raised against human chorionic gonadotropin. Serum concentrations of LH as well as estradiol, progesterone, and 17 alpha-hydroxyprogesterone up to day 6 after mating were determined in pregnant rabbits and compared with those in pseudopregnant and normal rabbits. In pregnant rabbits after mating, a surge of 62 +/- 15 ng of luteinizing hormone and 73 +/- 22 ng of progesterone/ml of serum occurred which returned to basal levels at 6 hours and on day 1, respectively. Secondary increases in serum luteinizing hormone of 26 +/- 12 to 36 +/- 16 ng/ml on days 3 and 5 and in serum progesterone of 16 +/- 4 and 14 +/- 5 ng/ml on days 5 and 6, respectively, were observed in pregnant rabbits.

A stimulatory effect of the fluid from preimplantation rabbit blastocysts upon luteinization of monkey granulosa cell cultures.

Blastocyst fluid was aspirated from Day 6 1/2--7 rabbit blastocysts and was added to cultures of granulosa cells obtained from preovulatory follicles of untreated rhesus monkeys or from follicles of monkeys or from follicles of monkeys treated with PMSG. The stimulation of progesterone secretion was measured and equated with that produced by hCG. The hCG-like activity was also measured in a radioreceptor assay using 125I-labelled hCG and porcine granulosa cells. In 8 out of 10 experiments with cultured cells from untreated monkeys, addition of 20% blastocyst fluid from Days 6--9 of culture stimulated progesterone secretion by 2- to 6-fold. Similar findings were obtained in 5 experiments with cultures from PMSG-treated monkeys except that the blastocyst fluid was added from Days 0 to 6 of culture. The granulosa cells in such cultures underwent morphological luteinization. Compared to a standard of purified hCG the blastocyst fluid contained about 0.76--2.5 ng hCG-like activity/ml which was non-dialysable. The radioreceptor assay indicated the presence of 0.5--2.5 ng hCG-like material/ml.

Implication of absence of HCG-like gonadotrophin in the blastocyst for control of corpus luteum function in pregnant rabbit

IN the primate, the corpus luteum formed after ovulation has a relatively short life span. If the ovum becomes fertilised, however, the presence of the embryo in the uterus leads to ‘rescue’ of the corpus luteum and a prolongation of its functional life1. Circumstantial evidence suggests that this is achieved through the elaboration of a luteotrophic substance (chorionic gonadotrophin) by the trophoblastic cells of the implanting embryo1,2. Based on differences in the plasma progesterone levels of pregnant and pseudopregnant animals, a similar situation has been proposed for the rabbit3. In support of this, the blastocyst fluid from 6-d pregnant rabbits has been reported to contain a substance similar to human chorionic gonadotrophin (HCG), by a radioreceptor assay4, and a substance similar to luteinising hormone (LH), by a heterologous radioimmunoassay5. Our study was initiated to identify definitively the source of the HCG-like substance in the rabbit blastocyst. Using a highly sensitive in vitro bioassay method we have examined the concentration of HCG-like material in the serum throughout pregnancy and in the blastocyst from days 4 to 7 of pregnancy. We report here that the rabbit blastocyst does not contain or secrete any gonadotrophin which has significant HCG-like activity.

Absence of maternal proteins in 5-7 day blastocyst fluid indicates limited protein passage before implantation.

AbstractRabbit serum albumin (RSA) and gamma globulin (RGG) were measured in fluid collected from single 5, 5.5, 6 and 6.5‐day blastocysts of New Zealand White rabbits, using a radial immunodiffusion method capable of detecting a minimum of 2–5 μg/ml in 1 μl. The samples showed little or no RSA and RGG. Three sources of contamination in fluid collection were identified. They are, blastocyst contact with aqueous solutions used to flush them out or rinse them, carrying in of surface proteins by pipette tips and withdrawal of trophoblast from lemmas. It is shown that fluid collected without precautions to avoid contamination will contain uterine fluid constituents.Bovine serum albumin (BSA), RSA and RGG pass readily into blastocyst fluid in vitro and after intrauterine injection. However, such passage is abnormal because BSA does not appear in 5–7‐day blastocyst fluid after intravenous injection of the mother (Kulangara and Crutchfield, '73) and normal 5–7‐day fluid shows little or no RSA and RGG. High viscosity and pH in the normal uterine lumen greatly reduce protein passage so that only traces reach trophoblast cells, which seem to digest this protein with lysosomal enzymes and/or transport it outward, thereby maintaining a virtually protein‐free blastocyst fluid, prior to implantation. Restriction of protein passage appears to be essential, since normal development is interrupted by intrauterine injections.

Uteroglobin and other proteins in rabbit blastocyst fluid after development in vivo and in vitro.

Rabbit embryos were grown in vitro from the 2- and 4-celled stage to expanded blastocysts. Proteins from the blastocysts were analyzed for specific uterine proteins as well as for bovine serum albumin (BSA), a constituent of the medium. Immunological methods revealed the presence of rabbit albumin and larger amounts of BSA. Uteroglobin, the prevailing protein fraction present in blastocyst fluid of embryos that developed in vivo was not detected in blastocysts in vitro. Prealbumin and beta-glycoprotein were also absent. From the data presented, it appears that the blastocyst does not have the capacity to synthesize detectable concentrations of uterglobin and/or other specific uterine proteins.