CN1863905A - Method and composition for enhancing in vitro embryo development by supplementing culture medium with prostaglandin or a prostaglandin analog - Google Patents

Abstract

A method of enhancing in vitro development of a mammalian embryo is disclosed which comprises supplementing the culture medium with a prostaglandin, or a prostaglandin analog, in an amount effective to promote complete hatching of the embryo (i.e., freeing of the embryo from the zona pellucida). Supplementation of the culture medium is preferably done during one or more of the following developmental stages of the embryo: (a) when the genome is activated and the fertilized egg develops into morulae; and (b) during the morulae to early blastocyst stage development of the embryo. The in vivo implantation potential and live birth potential of an in vitro fertilization embryo is thereby enhanced and establishment of a viable pregnancy is facilitated.

Description

Methods and compositions for enhancing in vitro embryonic development by supplementing culture medium with prostaglandins or prostaglandin analogs

Statement of federally funded research or development

This invention was made in whole or in part with funding from the National Institute of Child Health and Human Development (NICHD), grant number HD01277. Accordingly, the US Government has certain rights in this invention.

Background of the invention

field of invention

The present invention relates to compositions and methods for culturing mammalian embryos in vitro and enhancing pregnancy after implantation of the cultured embryos into the uterus of a suitable mammalian host.

Description of prior art

The environment within the fallopian tubes enhances the fertilization potential of the semen and promotes the development of cleaved embryos. Co-culture of embryos with oviduct epithelial cells has been shown to enhance development, hatching, and implantation [Xu, 2001; Xu, 2000].

It is traditionally believed that prostacyclin (PGI ₂ ) is related to the maintenance of blood and blood vessel homeostasis. However, recent observations of knockout mice suggest that they may have other physiological functions. The importance of endometrium-derived PGI ₂ is emphasized by observations in cyclooxygenase (COX)-2 knockout mice [Lim, 1997]. The endometrium of COX-2 knockout mice usually fails to shed. As a result, transferred wild-type embryos failed to implant in COX-2-deficient female mice. The abnormalities described above can be corrected to some extent by administering exogenous PGI ₂ analogues to the mother [Lim, 1999].

The role of oviduct-derived PGI ₂ in preimplantation development is unclear because litter sizes of PGI ₂ receptor (IP) knockout mice have not been compared to wild-type litter sizes [Murata , 1997]. However, the genotype distribution of pups from mated heterozygous IP knockout mice failed to follow Mendelian inheritance laws: the probability of male and female pups having homozygous IP knockout genotypes was 37% and 20%, respectively, less than expected [Murata, 1997].

Summary of the invention

Recently, we found that the human oviduct synthesizes a large amount of prostacyclin (PGI ₂ ) as well as PGE ₂ [Huang, 2002]. Knockout studies have shown that endometrium-derived PGI ₂ is essential for endometrial shedding, but the effects of PGI ₂ on preimplantation development have not been reported. Utilizing the hypothesis that PGI ₂ may be required for optimal preimplantation embryo development, the effects of PGI ₂ on mouse embryos were studied based on complete hatchability. Expression of PGI ₂ receptor (IP) was evaluated by Western blot analysis and immunohistochemistry. Binding of PGI ₂ to embryos was confirmed by radioligand binding assay. Our results demonstrate that the hatching of mouse embryos is enhanced by the addition of iloprost ( _ED50 6.7nM), a stable synthetic analog of _PGI2 . Exposure to iloprost at the 8-cell to morula or morula to early blastocyst stage was important for enhanced hatching. This stage-specific response to PGI ₂ is consistent with developmental stage-specific expression of IP. Further studies were performed in an attempt to link enhanced hatching of embryos to increased implantation and live birth rates by transferring mouse embryos cultured in Iloprost-supplemented medium into fertile vectors. The number of pregnant embryo sacs and viable pups was compared to the number of control embryos. Because supplements in the medium have been reported to increase fetal weight [DeBaun, 2002; Thompson, 1995; Sinclair, 1999], pup and placental weights were also compared. Our results show that culturing embryos in medium supplemented with iloprost enhanced mouse embryo implantation and live birth rates without affecting pup or placental weight.

Accordingly, in a particular embodiment of the invention there is provided a method of enhancing in vitro development of an embryo comprising supplementing the culture medium with a prostaglandin such as PGI ₂ or PGE ₂ or analogs thereof. In certain embodiments, the prostaglandin is PGI ₂ or PGE ₂ , and in certain embodiments the prostaglandin analog is iloprost or PGE ₁ . In certain embodiments, the method comprises adding to the culture medium a prostaglandin or the like in an amount sufficient to promote complete hatching of the embryo. Preferably, "full hatching" includes releasing the embryo from the zona pellucida. In certain embodiments of the method, the medium is supplemented during the early developmental stages of the embryo, when the genome is activated and the fertilized egg develops into a morula. In some embodiments where the embryo is human, the early developmental stages begin at the 4- to 8-cell state. In certain method embodiments, the medium is supplemented during the development of the embryo from the morula to early blastocyst stages. In certain method embodiments, the medium is supplemented during the early developmental stages of the embryo to the morula stage and during the development of the morula to early blastocyst stage of the embryo. In certain method embodiments wherein the embryos are of mouse origin, the supplementing step comprises exposing the embryos in culture to a prostaglandin or the like between 24 and 42 hours after harvest of the embryos at their 2-cell developmental stage. In certain method embodiments in which the embryo is a mouse, the supplementary step during the morula to early blastocyst stage comprises exposing the embryo to a prostaglandin between 42 and 72 hours after harvest of the embryo at its 2-cell developmental stage or prostaglandin analogues.

Certain embodiments according to the present invention also provide methods of increasing the in vivo implantation potential of in vitro fertilized embryos. "Implantation potential" is the ability of an embryo to implant into the uterus. The method comprises carrying out one of the above-mentioned embodiments to enhance the in vitro development of embryos, thereby obtaining complete hatching of embryos in culture or enhanced hatching compared to other IVF methods. According to certain embodiments of the method, the fully or nearly hatched embryos are then introduced into the uterus of the mammalian host, thereby obtaining enhanced implantation of the embryos. In certain embodiments, complete in vitro hatching of embryos is associated with the establishment of a viable pregnancy.

In certain embodiments of the invention, methods of increasing the live birth potential of in vitro fertilized mammalian embryos are provided. "Live birth potential" is the ability of an embryo to achieve live birth. The method comprises enhancing the in vitro development of the embryo by in vitro culture with an appropriate amount of a prostaglandin or an analog thereof as described above, thereby obtaining enhanced hatching potential or full hatching of the embryo in culture. In preferred embodiments, this treatment confers an enhanced potential for complete hatching, even after embryo removal from culture and in an in vivo environment. This trait is called "Enhanced Hatching Potential". The hatched embryos, or embryos with enhanced hatching potential, are then transferred into the uterus of a mammalian host; the embryos are allowed to implant and grow in vivo, thereby conferring on the embryos the ability to give birth to live relative to implanted embryos that have not been enhanced for hatching be strengthened.

In another embodiment of the present invention, there is provided a method of increasing the birth rate of a population of mammalian embryos fertilized in vitro, which comprises enhancing the in vitro development of embryos according to the method described above, so that hatching is enhanced, or preferably, the complete development of cultured embryos is obtained. incubation. The method comprises introducing or transferring embryos into a mammalian host; and then growing the embryos in vivo, wherein the live birth rate of the group of embryos having enhanced hatching is greater than the live birth rate of the group of embryos transferred into the uterus of the host, without first fully hatching or Receive hatching enhancements.

Certain embodiments of the present invention further provide improved cell culture media for in vitro mammalian embryo development, wherein the improvement comprises supplementation with prostaglandins or analogs thereof in an amount effective to enhance hatching, and preferably promote complete hatching of embryos in vitro. In certain embodiments, the medium contains a prostaglandin, or an analog thereof, effective to enhance the full hatching potential of the embryo after removal from in vitro culture and transfer into the uterus of a mammalian host. These and other embodiments, features and advantages of the invention will become apparent with reference to the following specification and drawings.

Description of drawings

Figure 1 is a graph showing prostacyclin ( _PGI2 ) and complete hatching of mouse embryos. 2-cell mouse embryos were cultured with PGI ₂ analogs (iloprost, 1 nM to 10 [mu]M). Complete hatching was determined after 96 hours. The results of 3 to 5 independent experiments (17-20 embryos per experiment) are expressed as mean ± SD. The _ED50 value is about 6.7nM.

Figure 2 is a bar graph showing the boost to full hatch and during prostacyclin ( _PGI2 ) exposure (iloprost exposure based on stage of embryo development). 2-cell mouse embryos were cultured in the presence of 0.1 [mu]M iloprost, a PGI ₂ analogue, during the indicated cycles (expressed as hours after harvesting 2-cell embryos). Complete hatching rates are expressed as mean ± SD. The developmental stages during which the embryos were exposed to iloprost are listed in the table. Two stages appear to be critical for the full effect of iloprost: 24-42 hours and 42-72 hours, corresponding to the development from 8-cell embryo to morula and morula to early blastocyst, respectively. Exposure to iloprost at various stages ensured enhancement of hatching. The number of independent observations (18-20 embryos each) were: control, 12; 42-72 hours, 3; others, 4. The complete hatching rate in control embryos was different from that in Figure 1, probably because of the change in the origin of the male mice.

Figure 3 is a Western blot showing prostacyclin receptor (IP) expression in mouse embryos. Western blot analysis of total cell lysates from 60 mouse blastocysts (lane 1 ) as well as microsomes from human platelets (positive control, lane 2) showed immunoreactivity with antibodies against human IP. Mouse IP migrates less than human IP, consistent with predicted molecular weights of about 50 kDa and about 46 kDa, respectively.

Figures 4A-E and G are photomicrographs showing expression of prostacyclin receptor (IP) in morulae and blastocysts. Phase contrast (FIG. 4A) and IP staining (FIG. 4B) visualization of 2 morulae are shown. The zona pellucida is shown by arrows. Figure 4C shows propidium iodide staining of nuclei in blastocysts. IP and propidium iodide staining of blastocysts in Figure 4D overlaid. Figure 4E shows IP staining of blastocysts, showing a reticular pattern. Clusters of IP staining were observed only in the trophectoderm; staining showing the inner cell mass was not enhanced. Figure 4F is a sketch showing the location of the inner cell mass (ICM) within the blastocyst. Figure 4G is a confocal microscopy image of a blastocyst showing only the trophectoderm stained by the IP antibody. Sections are shown by dashed lines in panel F. Negative controls, no IP staining in unfertilized oocytes or embryos at other developmental stages (not shown). The lines in the graph are about 20 μm.

Figure 5 is a bar graph showing iloprost binding to intact mouse embryos. 116 mouse blastocysts were incubated with 0.1 μM ³ H-iloprost, PGI ₂ analog in the presence or absence of 5 μM unlabeled iloprost. Bound as well as free ³ H-iloprost were separated by membrane filtration.

Figures 6A-B show a pregnant embryo sac in the uterus of an impregnated carrier. Implantation was determined by increased vascular supply to the implantation site and confirmed by the presence of a pregnant embryo sac. Seventy-two hours after embryo transfer, conceived vectors were administered 1% Chicago blue via the tail vein and euthanized 3 minutes later. Figure 6A shows a discontinuous blue band (indicated by *) surrounding the uterine horn of the recipient embryo. In Figure 6B, each blue band corresponds to a pregnant embryo sac, which appears as pink tissue in the open uterine horns. No blue bands or pregnant embryo sacs were found in the uterine horns (indicated by arrows) that did not receive embryos.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present disclosure was developed during the course of studies of the effects of PGI ₂ and PGE ₂ on semen and embryos, and subsequent investigations. Our recent results showed that human [Huang, 2002] and mouse [Huang, 2004] oviduct epithelial cells express the enzymes necessary for PGI ₂ synthesis, namely COX-1 or COX-2 and PGI ₂ synthetase. Large amounts of PGI ₂ and PGE ₂ were produced when ¹⁴ C-arachidonic acid was incubated with microsomes prepared from human [Huang, 2002] or mouse oviduct [Huang, 2004]. This study also takes into account the fact that semen travels to the distal fallopian tube to fertilize the egg and the first 72 hours of embryonic development occur in the fallopian tube.

As part of this study, the effect of PGI ₂ on embryos was evaluated by observing complete hatching of mouse embryos cultured with PGI ₂ analogs. To further elucidate the mechanism, IP expression and embryo binding were also studied by radiolabeled iloprost.

Materials and Methods

Source of reagents and licensing of the system.

Unless otherwise stated, reagents were purchased from Sigma Co. (St. Louis, MO, USA). The use of human samples was approved by the Committee for the Protection of Human Subjects (equivalent to an institutional review committee). The management of experimental animals and the research protocol were approved by the Animal Welfare Committee.

Mouse embryos were harvested and cultured.

Mice were housed with free access to water and food under conditions of controlled temperature, humidity and light cycle (12 hours light/dark). 3-week-old C57B1/6 female mice were purchased from Harlan (Indianapolis, IN, USA). 8-week-old C3H male mice were purchased initially by Harlan and later by The Jackson Laboratory (Bar Harbor, ME, USA). Superovulation in female mice was achieved by intraperitoneal injection of pregnant horse serum gonadotropin (5 IU) followed by human chorionic gonadotropin (hCG, 5 IU) 46 hours later. After receiving hCG, each female mouse was paired with a fertile male mouse. After 48 hours, 2-cell embryos were harvested from the oviduct into α-MEM medium supplemented with 25 mM HEPES and 1% BSA (Irvine Scientific).

Embryos (17-20 per group) were cultured in 4-well plates (NalgeNunc International, Naperville, IL, USA) containing 600 μl of medium per well at 37° C., 5% CO ₂ . HTF and α-MEM medium were used sequentially over a 96-hour period to match the changing nutritional requirements of lysed embryos [Gardner, 1998]. HTF medium (SAGE Biopharma) was used during the first 48 hours, and α-MEM medium (Irvine Scientific) with Earle's salts and 2 mM glutamine was used during the second 48 hours. Experimental embryos received iloprost in water and control embryos received an equal amount of water. Preliminary experiments showed that more than 95% of the embryos became blastocysts by 96 hours, similar to embryos cultured in KSOM medium (Specialty Media, Cell and Molecular Technologies, Inc. Phillipsburg, NJ, USA). After 96-hour culture, each embryo was checked for the presence of zona pellucida. Embryos completely free of zona pellucida were counted as fully hatched embryos. The complete hatch rate was determined by dividing the fully hatched embryos by the total number of embryos. Complete hatching of embryos was chosen as the endpoint rather than blastocyst formation or embryo hatching, as the latter 2 markers were not associated with the establishment of a viable pregnancy [Lane, 1997].

Western blot analysis

The deduced amino acid sequences of mouse IP (417a.a., Genebank BAA05144) and human IP (386 a.a., Genebank_BAA06110) are highly homologous [Katsuyama, 1994]. Preliminary studies demonstrate that affinity-purified polyclonal peptide antibodies (kindly provided by Dr. Ke-He Ruan, University of Texas Health Science Center) cross-react with mouse IP. Western blot analysis was performed as previously described [Huang, 2002]. Briefly, total cell lysates from 60 mouse blastocysts were separated by separation on 10% acrylamide gel (PAGE) and transferred to nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH, USA). Immunoactive proteins were detected by incubation with antibodies and visualization with enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA) using a STORM 860 laser scanner (Amersham Biosciences). Human platelet microsomes were used as a positive control. Antibody specificity was confirmed in parallel experiments using preabsorbed antibodies.

Immunohistochemistry, fluorescence microscopy, and confocal microscopy.

Mouse embryos were fixed in 4% paraformaldehyde (pH 7.4) for 30 minutes at 4°C. After washing with PBS for 3 times, the embryos were blocked with Tris-buffered salt containing 0.05% Tween-20, 5% milk powder and 0.1% Triton X-100 for 20 minutes at room temperature. Embryos were incubated with blocking buffer of IP antibody (5ng/ml) for 2 hours, and then incubated with Alexa 488-coupled goat anti-rabbit IgG (2.5 μg/ml, Molecular Probes, Eugene, OR, USA) at 37°C for 30 minute. Nuclei were counterstained with 10 μg/ml propidium iodide for 20 min at room temperature. Embryos were mounted on Fluoromount-G ^(R) (Southern Biotechnology Associates Inc., Birmingham, AL, USA). For fluorescence microscopy, place blastocysts in mounting medium and cover with coverslips. For confocal microscopy, a spacer of approximately 50 μm was placed between the glass slide and the coverslip to maintain the three-dimensional morphology of the embryos. For negative controls, embryos were incubated with 10 ng/ml of non-immune rabbit IgG (ie, twice the concentration of the primary antibody).

Fluorescence microscopy was performed using a Zeiss AxioPlan 2 microscope equipped with appropriate filters (Carl Zeiss, Baden-Wuerttemberg, Germany). Images were captured with a CCD camera and processed by the AxioVision program (version 3.0.6). Confocal microscopy was performed using a BioRad Radiance 2000 confocal system (Bio-Rad Laboratories, Hercules, CA, USA) attached to an Olympus BX-50 microscope. Images were processed using the Image-Pro ⁺ program (Media-Cybernetics, Carlsbad, CA, USA).

Whole Embryo Radioligand Binding Assay

Binding assays ^of3H -iloprost to blastocysts were performed as previously described with only slight modifications [Arbab, 2002]. 232 hatched and hatching blastocysts were washed with binding buffer (10 mM _MnCl2 in 10 mM HEPES, pH 7.4) and then transferred to 100 μl of binding buffer with or without unlabeled iloprost (5 μM). The reaction was initiated by adding an equal amount of binding buffer containing 3H-iloprost (200 nM, specific activity 11.0 Ci/mmol, Amersham Biosciences) and incubated at room temperature for 60 minutes. The reaction was terminated by transferring the blastocysts to 2 ml of ice-cold wash buffer (0.01% BSA in 10 mM HEPES, pH 7.4). The buffer and blastocysts were filtered through a glass fiber filter (Whatman GF/C 2.4 cm) and the filter was washed 3 times with 2 ml of wash buffer. Filtrates were dried in an oven before radioactivity was determined by scintillation counting with 5 ml of scintillation liquid.

Statistical Analysis

Where appropriate, Student's t-test or one-way analysis of variance followed by Dunnett's test was used. p<0.05 was considered statistically significant. Construction of dose-response curves and calculation of _ED50 values (curve slope set at 1.0) was performed by GraphPad Prism ^(R) software (GraphPad PrismSoftware Inc., San Diego, CA, USA).

embryo transfer

Mouse embryos were harvested and cultured as described above and subsequently disclosed by the present inventors (Huang et al., 2003). Briefly, 2-cell embryos (C3B6F1 ) were harvested from superovulated 3-week-old C57B1/6 female mice 42 hours after hCG injection. Embryos were cultured in 4-microwell plates (Nalge Nunc International, Naperville, IL, USA) each containing 600 μl of HTF medium (SAGE Biopharma, Bedminster, NJ, USA) at 37°C under 5% _CO conditions (per group of 14). Experimental embryos received iloprost (1 [mu]M, Caymen Chemical, Ann Arbor, MI, USA) in water and control embryos received an equivalent amount of water. In both cases, the volume of water was less than 0.1% of the medium.

The day after embryo donors received hCG, vaginal smears were obtained from potential pregnant carriers (8-week-old C3B6F1 female mice) to determine the stage of the estrous cycle. Those animals in heat were paired with vasectomized ICR male mice (Harlan) and the vaginal tethers were checked the following morning. Animals with a vaginal tether were referred to as 0.5 day pseudopregnant.

Embryo transfer was performed on day 2.5 of pseudopregnancy under a dissecting microscope (Olympus SZ-PT). Surgical anesthesia was obtained by intraperitoneal injection of ketamine (200 mg/Kg) and xylazine (10 mg/Kg) (both obtained from Burns Vet Supply Inc. TX, USA). Each uterine horn was accessed through a 2-cm lateral incision. The adjacent fallopian tubes are secured with a pair of forceps, and an opening is created on the anti-mesenteric side with a 30-gauge needle distal to the uterine horn. This opening allows the entry of a pipette pipette with an inner diameter of 135 μm (MidAtlantic Diagnostics, Inc. NJ, USA). Up to 7 embryos were transferred into each horn in 0.8 μl of transfer medium (αMEM with 25 mM HEPES and 1% BSA). After each transfer, the contents of the pipettes were examined under a stereomicroscope to identify retained embryos. The flank incisions were closed with 9 mm metal clips (Clay Adams, Parsippany, NJ, USA). Since the embryos migrated from one corner to the other (personal contact with Dr. Andreas Zimmer, University of Bonn, Germany), in order to avoid mixing of control and experimental embryos, each conception carrier received either control or experimental embryos. In order to maintain a consistent transfer technique, embryo transfer was performed according to the same protocol and by the same individual who was blinded to the treatment they were scheduled to receive. In order to ensure that both groups benefited equally from the experience gained during the study (approximately 6 months), embryos were assigned treatments (control or iloprost) in 4 blocks.

Determine implantation rate

Seventy-two hours after embryo transfer, implantation was determined according to the method described previously (Paria et al., 1993) with some modifications. Briefly, 0.1 ml of Chicago blue (1%) was injected into the tail vein of the impregnated vehicle 3 minutes before euthanasia. After sacrifice of the vehicle, the uterine horns were removed. Discrete blue bands (shown as *) surrounding the uterine horns were counted (Figure 6A). The presence of these bands reflects the enhancement of the vascular supply of the blood vessels at the implantation site. The uterine horns were then opened and the pregnant embryo sacs counted (Figure 6B). Implantation rates are expressed as the number of embryo sacs per embryo transferred.

Determination of birth rate and weight of pups and placenta

Preliminary studies have shown that some conceived carriers eat their young when their litter size is small. In order to accurately count and weigh the pups, we sacrificed the conceived carriers 14 days after embryo transfer (16.5 days of gestation or 2 days before natural birth). Following euthanasia, live pups were counted and individual pup and placental weights determined. Check all pups for gross abnormalities. The number of empty pregnant embryo sacs was also counted to determine the implantation rate. Birth rate is expressed as the number of live pups per embryo transferred; implantation rate is expressed as the number of embryo sacs with or without live pups per embryo transferred.

Statistical Analysis

Fisher's exact test was used to compare proportions; Student's t-test was used to compare weights. p<0.05 was considered statistically significant. GraphPad Instal ^(R) software (GraphPadSoftware Inc., San Diego, CA, USA) was used for statistical analysis.

result

PGI ₂ improves complete hatching of mouse embryos.

Complete hatching of mouse embryos was enhanced by iloprost in a concentration-dependent manner. At concentrations of 0.1 [mu]M or higher, the effect was statistically significant (Figure 1). The greatest enhancement of complete embryo hatching occurred at 1 μM, where 81±7% (mean±SDn=3) of experimental embryos hatched completely. In contrast, only 49±14% (mean±SDn=5) of control embryos fully hatched. The _ED50 value derived from the dose response curve was 6.7 nM (Figure 1). A saturating concentration-dependent response and an _ED50 value of 6.7 nM indicated that the effect of iloprost was receptor mediated.

PGI ₂ exposure time and stage of embryo development critical for enhanced hatching.

Because the embryo goes through several developmental stages before entering the uterus, we investigated the developmental stage-specific response to iloprost. Our results show that some times during the 96-hour culture period are more important than others. Exposure to iloprost during the initial 24-hour culture period (during which most 2-cell embryos develop into 8-cell embryos) did not enhance hatching (Figure 2). On the other hand, exposure to iloprost between 0-72 hours or 24-72 hours post-harvest resulted in the same complete hatch rate as exposure between 0-96 hours (Figure 2).

The 2 critical times of exposure to iloprost to ensure the full effect of iloprost are between 24 to 42 hours and 42 to 72 hours after harvest, corresponding to the transition from 8-cell embryos to morula and from morula to Transformation of embryos to early blastocysts (Figure 2 bottom panel). Our data show that brief exposure of embryos to PGI ₂ enhances the potential for full hatching, even after embryos exit the oviduct.

Mouse embryos express IP.

To study developmental stage-specific expression of IP in mouse embryos, we performed Western blot analysis on blastocysts and immunohistochemical analysis on embryos at different developmental stages. Western blot analysis showed detection of protein of expected molecular weight by affinity-purified anti-IP antibody (Figure 3). Fluorescence microscopy revealed IP staining in morulae and blastocysts (Fig. 4A-E) but not in unfertilized eggs, or in 1-cell, 2-cell, 4-cell, and 8-cell embryos (not shown) . IP staining in morula as well as in blastocysts had the same fine network pattern. Confocal microscopy visualization revealed preferential expression of IP in trophectoderm (Fig. 4G). Thus, embryonic stage-specific expression of IP by mice is consistent with a response to iloprost.

Radiolabeled iloprost binds to mouse embryos.

The concentration-dependent enhancement of complete embryo hatching by iloprost showed that the effect was receptor mediated. We performed radioligand binding assays to further confirm that mouse embryos are bound by iloprost. We chose to quantify iloprost binding at the ligand level of 0.1 μΜ where hatch enhancement was first observed and found that approximately 3.04 fmol ^of3H -iloprost bound specifically to 116 blastocysts (Figure 5).

discuss

Our data show for the first time that PGI ₂ enhances complete hatching of embryos. This is consistent with previous reports that co-culture of mouse embryos with human oviduct epithelial cells increased embryo cell numbers, decreased apoptosis, and improved embryo hatching [Piekos, 1995; Xu, 2000; Xu, 2001]. Thus, fallopian tube-derived PGI ₂ as well as vascular endothelium-derived PGI ₂ act in a paracrine-like manner. The former enhances embryo hatching; the latter prevents platelet aggregation. A concentration-dependent response was consistent with a receptor-mediated event and the _ED50 value of 6.7 nM was similar to the Kd value reported for lysed IP (8 nM) from human platelets [Tsai, 1989].

Embryo responses to iloprost were developmental stage specific and consistent with IP expression (Figure 2). These critical times of response coincide with the sojourn of the mouse embryo in the oviduct, during which time the fertilized egg develops into a morula. Associated developmental stages also coincide with genome activation in human and mouse embryos, which occur between the 4- and 8-cell stages and after the two-cell stage, respectively [Tesarik, 1986; Tesarik, 1988; Braude, 1988].

Without wishing to be bound by a particular theory of how IP exerts its effects, we propose that embryos exposed to oviduct-derived _PGI2 or _PGE2 early in development maintain enhanced hatching potential as they reach the uterus. Via the oviduct-derived PGI ₂ , the embryos actively prepare for their own implantation while they are still in the oviduct. From an embryonic perspective, oviduct-derived PGI ₂ complements endometrium-derived PGI ₂ , which mediates shedding of the endometrium to ensure receptivity [Lim, 1999].

Without wishing to be bound by a particular theory, we propose that enhanced hatching mediated by PGI ₂ or PGE ₂ is attributable to an increase in the number of blast cells. Different mechanisms involve hatching in vitro as well as in vivo in mouse embryos. In vitro hatching involves blastocyst expansion leading to total zona thinning prior to zona rupture, whereas in vivo hatching involves comprehensive zona lysis by uterine or trophectoderm cytolysins [Montag, 2000]. Therefore, a sufficiently high number of blast cells is required to complete in vitro incubation. In this regard, our results are consistent with the findings that co-cultures of embryos with oviduct epithelial cells had more cells, less cell death, and improved hatching [Yeung, 1992; Xu, 2000]. In addition, PGI ₂ can enhance the production of trypsin-like proteases from the trophectoderm to dissolve the zone [Sawada, 1990; Perona, 1986].

The low implantation potential of IVF embryos (10-20% per embryo) [CDC, 2001] is partly due to suboptimal culture conditions [De Vos, 2000]. Media have been modified to enhance in vitro embryo development [Gardner, 1998]. According to our data, medium supplemented with PGI ₂ and/or PGE ₂ analogues such as iloprost, PGE ₁ can provide a modified environment for embryo development and increase its implantation potential.

Finally, PGI ₂ enhances complete hatching of cultured mouse embryos. Embryo stage-specific expression of IP by mice is consistent with its response to PGI ₂ . Finally, according to our initial data, PGE ₂ also enhances complete hatching of cultured mouse embryos.

In a further study, we demonstrated that medium supplemented with PGI ₂ enhanced mouse embryo implantation and potentially live birth. These results confirmed our initial observation of complete hatching of mouse embryos in PGI ₂ boosted cultures [Huang, 2003]; they also supported our hypothesis that oviduct-derived PGI ₂ could provide physiological functions [Huang, 2002].

Table 1 shows that PGI ₂ enhances the implantation potential of mouse embryos. 84 control embryos as well as 81 iloprost-treated embryos were transferred into 12 fertile vectors. After 72 hours, more pregnant embryo sacs were found in the iloprost-treated group than in the control group (76% vs. 42%, relative risk 1.84, 95% confidence interval 1.38-2.43, p<0.0001). At the time of transfer, the developmental stages of the embryos can be compared.

Table 2 shows that PGI ₂ iloprost enhanced the live birth potential of mouse embryos. 406 control embryos and 415 iloprost-treated embryos were transferred into 30 and 31 pregnant vectors, respectively. The birth rates in the control and experimental groups were 28% and 36%, respectively (p=0.0017, relative risk 1.28, 95% confidence interval: 1.044-1.560). Pups as well as placenta weights were comparable. Thus, PGI ₂ increased the live birth potential of mouse embryos without affecting pup or placental weight. Additionally, there are no gross anomalies recorded.

As noted above, approximately 70% of experimental as well as control embryos were at the morula stage at the time of transfer (Table 1), however transferred experimental embryos produced more pregnant embryo sacs. These results confirm our previous observation that exposure to PGI ₂ between the 8-cell and morula stages is sufficient to enhance complete hatching in mice [Huang, 2003]. They are also consistent with oviduct-derived PGI ₂ as an embryotrophic factor, as fertilized mouse eggs develop into morulae within the oviduct [Snell, 1966].

These observations suggest that PGI ₂ initiates a cascade of events that ultimately enhances hatching, implantation and increases live birth rates. In this regard, PGI ₂ is reminiscent of platelet activating factor (PAF), with short exposure to PAF enhancing implantation in mice [O'Neill, 1998] as well as in human embryos [O'Neill, 1989]. Molecular as well as cellular events initiated by PGI ₂ are important for incubation. They may include increased blast cell numbers [Montag, 2000], enhanced production of trypsin-like proteases by the trophectoderm [Sawada, 1990; Perona, 1986], enhanced Na ⁺ -K ⁺ -ATPases due to enhanced trophectoderm Systematic blastocoel expansion [Biggers, 1988] or a mechanism yet to be discovered.

Implantation rates of control embryos (42% and 46%) were compared when examined at 72 hours or 14 days after transfer, respectively. On the other hand, the implantation rate of the experimental embryos examined 14 days after transfer (59%) was significantly lower (p=0.038) than that tested 72 hours after transfer (76%). It is plausible that more embryo sacs were fully resorbed in the experimental group due to the aggregation of implantation sites. Alternatively, iloprost "rescues" some embryos that may not implant and only a fraction of those "rescued" embryos develop into live pups. The results of this study and the production of large amounts of PGI ₂ by human [Huang, 2002] as well as mouse [Huang, 2004] oviducts show that PGI ₂ is an embryotrophic factor secreted by the oviduct, and that embryo culture is therefore supplemented with PGI ₂ analogues. The base should improve IVF outcomes.

Supplementation of IVF media with substances that enhance embryo development or implantation has been adopted by most laboratories and by careful media manufacturers. Possible concerns include issues regarding reproductive toxicology, teratogenicity, and possible exogenous effects of such manipulations. Lambs from sheep embryos cultured in medium supplemented with human serum are heavier and have longer gestation periods [Thompson, 1995; Holm, 1996; Sinclair, 1999]. The congenital disorder of Beckwith-Wiedermann syndrome, including overgrowth and tumor formation, has been associated with assisted regenerative techniques [DeBaun, 2002; Maher, 2003].

Reproductive toxicology studies on iloprost [Battenfeld, 1995] showed that embryonic and fetal development was not affected in rabbits and monkeys, but a reduction in digits was observed in rats. The toe abnormalities may be due to reduced uteroplacental fluidity causing hypoxia of the affected structures rather than congenital teratogenicity of iloprost, which is a vasodilator and was administered throughout gestation in animals studied iloprost. Similar deficits can be induced by vasodilators with different chemical structures such as hydralazine and dihydropyridine. In the current study, exposure to iloprost occurred during the preimplantation period. Of the 246 live pups examined, there were no digit reductions or other abnormalities and pup and placental weights were similar between the 2 groups. Nonetheless, supplementation of IVF medium with PGI ₂ analogs should be considered an experimental protocol.

In these exemplary studies, we demonstrate that PGI ₂ enhances implantation and live birth potential of mouse embryos without affecting fetal or placental weight, ie, prostacyclin does not aggravate placenta or infant. This is an advantage independent of implantation and live birth enhancement, and shows that prostacyclin is different from other agents that enhance implantation and live birth. Agents previously reported to enhance implantation and live birth also aggravate the infant, rendering those agents unsuitable for in vitro fertilization (IVF) applications in humans. Furthermore, since prostacyclin does not affect the placenta as well as infant weight in an in vivo mouse model, it could be considered for use in human IVF embryos.

The above in vivo results obtained with mouse embryos are considered representative or predictive of similar favorable in vivo results that may be obtained in embryos of other mammals, including humans, when those embryos are similarly treated with prostaglandins or Prostaglandin treatment in vitro. Current human IVF involves the transfer of either 8-cell stage or blastocyst stage embryos to the patient's uterus. In this regard, human IVF is similar to our experimental design involving the transfer of control or prostacyclin analog-treated embryos to fertilized vehicles. Our data showed that experimental embryos exposed to prostacyclin analogs between the 8-cell and morula stages had significantly higher implantation and live birth rates than control embryos when transferred into fertile carriers (Table 1, respectively). and 2).

Table 1 deal with P control iloprost total transferred embryos 84 81 degenerated 0 0 ns Morula 48 49 ns early blastocyst+ 12 8 ns late blastocyst+ twenty four twenty two ns hatching blastocyst 0 2 ns number of embryo sacs 35 (42%) 62 (76%) ＜0.0001 ^*

n.s. not significant

^* Fisher's exact test, associated risk 1.84 (95% confidence interval: 1.39-2.43)

+Early and late blastocysts represent embryos that occupy <49% and ≥50% of the blastocoel, respectively.

Table 2 deal with P control iloprost total transferred embryos 406 415 Number of pregnant carriers 30 31 Total number of live pups 115 (28%) 149 (36%) 0.017 ^* Pup Weight (gm ^** ) 0.788±0.151 0.772±0.124 ns Placenta Weight (gm ^** ) 0.166±0.037 0.166±0.003 ns

n.s. not significant

^* Fisher's exact test, associated risk 1.28 (95% confidence interval: 1.04-1.56)

^** Expressed as mean ± SD.

1. Arbab, F., Goldsby, J., Matijevic-Aleksic, N., Huang, G., Ruan, K.H., and Huang, J.C. (2002) Prostacyclin is an autocrine regulator in the contraction of oviductal smoothmuscle. Hum Reprod. 17, 3053-9.

2. Battenfeld, R., Schuh, W., and Schobel, C. (1995) Studies on reproductive toxicity ofiloprost in rats, rabbits and monkeys. Toxicol Lett.78, 223-34.

3. Biggers, J.D., Bell, J.E., and Benos, D.J. (1988) Mammalian blastocyst: transport functions in a developing epithelium. Am J Physiol.255, C419-32.

4. Braude, P., Bolton, V., and Moore, S. (1988) Human gene expression first occurs between the four-and eight-cell stages of preimplantation development. Nature. 332, 459-61.

5. CDC. (2001). "ART Cycles Using Fresh, Nondonor Eggs or Embryos." 1999 Assisted Reproductive Technology Success Rates, National Summary and Fertility Clinic Reports, U.S. Department of Health and Human Services Center for Prevention3 and Disease, Control5.

6. De Vos, A., and Van Steirteghem, A. (2000) Zona hardening, zona drilling and assisted hatching: new achievements in assisted reproduction. Cells Tissues Organs. 166, 220-7.

7. DeBaun, M.R., Niemitz, E.L., and Feinberg, A.P. (2002) Association of In VitroFeltilization with Beckwith-Wiedemann Syndrome and Epigenetic Alterations of LIT1and H19. Am J Hum Genet.72, 1.

8. Gardner, D.K. (1998) Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryoculture. Theriogenology. 49, 83-102.

9. Holm, P., Walker, S.K., and Seamark, R.F. (1996) Embryo viability, duration ofgestation and birth weight in sheep after transfer of in vitro matured and in vitro fertilized zygotes cultured in vitro or in vivo.J Reprod 0 Fertil , 175-81.

10. Huang, J.C., Arbab, F., Tumbusch, K.J., Goldsby, J.S., Matijevic-Aleksic, N., and Wu, K.K. (2002) Human Fallopian Tubes Express Prostacyclin (PGI) Synthase and Cyclooxygenases and Synthesize Abundant PGI.J Clin Endocrinol Metab. 87, 4361-4368.

11. Huang, J.C., Goldsby, J.S., Arbab, F., Melham, Z., Alecsic, N., and Wu, K.K. (2004) Oviduct Prostacyclin Functions as a Paracrine Factor to Augment the Development of Embryos.Hum Reprod.in press .

12. Huang, J.-C., Wun, W.-S.A., Goldsby, J.S., Wun, I.C., Falconi, S.M., and Wu, K.K. (2003) Prostacyclin enhances embryo hatching but not sperm motility. Hum. Reprod., 18, 2582-2589.

13. Katsuyama, M., Sugimoto, Y., Namba, T., Irie, A., Negishi, M., Narumiya, S., and Ichikawa, A. (1994) Cloning and expression of a cDNA for the human prostacyclin receptor. FEBS Lett. 344, 74-8.

14. Lane, M., and Gardner, D.K. (1997) Differential regulation of mouse embryodevelopment and viability by amino acids. J Reprod Fertil. 109, 153-64.

15. Lim, H., Gupta, R.A., Ma, W.G., Paria, B.C., Moller, D.E., Morrow, J.D., DuBois, R.N., Trzaskos, J.M., and Dey, S.K. (1999) Cyclo-oxygenase-2-derived prostacyclinmediates embryo implantation in the mouse via PPARdelta. Genes Dev.13, 1561-74.

16. Lim, H., Paria, B.C., Das, S.K., Dinchuk, J.E., Langenbach, R., Trzaskos, J.M., and Dey, S.K. (1997) Multiple female reproductive failures in cyclooxygenase 2-deficientmice. Cell.91, 197- 208.

17. Maher, E.R., Brueton, L.A., Bowdin, S.C., Luharia, A., Cooper, W., Cole, T.R., Macdonald, F., Sampson, J.R., Barratt, C.L., Reik, W., and Hawkins, M.M. (2003) Beckwith-Wiedemann syndrome and assisted reproduction technology (ART). J Med Genet. 40, 62-64.

18.Montag, M., Koll, B., Holmes, P., and van der Ven, H. (2000) Signficance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts invitro versus in vivo. Biol Reprod. 62, 1738-44.

19. Murata, T., Ushikubi, F., Matsuoka, T., Hirata, M., Yamasaki, A., Sugimoto, Y., Ichikawa, A., Aze, Y., Tanaka, T., Yoshida, N. ., Ueno, A., Oh-ishi, S., and Narumiya, S. (1997) Altered pain perception and inflammatory response in mice lacking prostacyclin receptor. Nature. 388, 678-82.

20. O'Neill, C. (1998) Autocrine mediators are required to act on the embryo by the 2-cellstage to promote normal development and survival of mouse preimplantation embryos invitro. Biol Reprod.58, 1303-9.

21. O'Neill, C., Ryan, J.P., Collier, M., Saunders, D.M., Ammit, A.J., and Pike, I.L. (1989) Supplementation of in-vitro fertilization culture medium with platelet activating factor. Lancet.2, 769-72.

22. Paria, B.C., Huet-Hudson, Y.M., and Dey, S.K. (1993) Blastocyst's state of activity determines the "window" of implantation in the receptive mouse uterus. Proc Natl AcadSci USA.90, 10159-62.

23. Perona, R.M., and Wassarman, P.M. (1986) Mouse blastocysts hatch in vitro by using atrypsin-like proteinase associated with cells of mural trophectoderm. Dev Biol. 114, 42-52.

24. Piekos, M.W., Frasor, J., Mack, S., Binor, Z., Soltes, B., Molo, M.W., Radwanska, E., and Rawlins, R.G. (1995) Evaluation of co-culture and alternative culture systems for promoting in-vitro development of mouse embryos. Hum Reprod. 10, 1486-91.

25. Sawada, H., Yamazaki, K., and Hoshi, M. (1990) Trypsin-like hatching protease from mouse embryos: evidence for the presence in culture medium and its enzymatic properties. J Exp Zool.254, 83-7.

26. Sinclair, K.D., McEvoy, T.G., Maxfield, E.K., Maltin, C.A., Young, L.E., Wilmut, I., Broadbent, P.J., and Robinson, J.J. (1999) Aberrant fetal growth and development after in vitro culture of sheep zygotes. J Reprod Fertil. 116, 177-86.

27. Snell, G.D., and Stevens, L.C.(1966). "Early embryology." Biology of the Laboratory Mouse by the staff of the Jackson Laboratory. E.L. Green, ed., Blakiston Division, McGraw-Hill Book Company, New York, 205 -245.

28. Tesarik, J., Kopecny, V., Plachot, M., and Mandelbaum, J. (1986) Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro. J Reprod Fertil. 78, 463 -70.

29. Tesarik, J., Kopecny, V., Plachot, M., and Mandelbaum, J. (1988) Early morphological signs of embryonic genome expression in human preimplantation development as revealed by quantitative electron microscopy. Dev Biol. 128, 15-20.

30. Thompson, J.G., Gardner, D.K., Pugh, P.A., McMillan, W.H., and Tervit, H.R. (1995) Lamb birth weight is affected by culture system utilized during in vitro pre-elongationdevelopment of ovine embryos. Biol Re3prod.53, -91.

31. Tsai, A.L., Hsu, M.J., Vijjeswarapu, H., and Wu, K.K. (1989) Solubilization of prostacyclin membrane receptors from human platelets. J Biol Chem.264, 61-7.

32. Xu, J., Cheung, T.M., Chan, S.T., Ho, P.C., and Yeung, W.S. (2000) Human oviductal cells reduce the incidence of apoptosis in cocultured mouse embryos. Fertil Steril.74, 1215-9.

33. Xu, J.S., Cheung, T.M., Chan, S.T., Ho, P.C., and Yeung, W.S. (2001) Temporal effect of human oviductal cell and its derived embryotrophic factors on mouse embryodevelopment. Biol Reprod.65, 1481-8.

34. Yeung, W.S., Ho, P.C., Lau, E.Y., and Chan, S.T. (1992) Improved development of human embryos in vitro by a human oviductal cell co-culture system. Hum Reprod.7, 1144-9.

Without further elaboration, it is believed that one skilled in the art can, using the description of the invention, utilize the present invention to its fullest extent. The above-described embodiments are illustrative and do not limit the remaining disclosure in any way. While preferred embodiments of the present invention have been shown and described, modifications can be made thereto by those skilled in the art without departing from the spirit and teachings of the invention. The embodiments described here are exemplary only, and not restrictive. Many variations and modifications can be made to the invention disclosed herein and are within the scope of the invention. References discussed in the Background of the Invention of the Invention are not prior art to the present invention, especially documents with publication dates prior to the priority date of the present invention. All patents, patent applications, and publications cited herein are incorporated herein by reference to provide exemplary, procedural, or other detailed supplements to the documents cited herein.

Claims (16)

1. A method for enhancing the in vitro development of mammalian embryos, comprising supplementing the culture medium with prostaglandins or analogs thereof in an amount effective to promote the complete hatching of embryos. 2. The method of claim 1, wherein the prostaglandin is prostacyclin ( _PGI2 ) or prostaglandin _E2 ( _PGE2 ). 3. The method of claim 1, wherein the prostaglandin analog is iloprost or prostaglandin _E1 ( _PGE1 ). 4. The method of claim 1, wherein said supplementation of the medium is performed during the early developmental stages of the embryo, during which the genome is activated and the fertilized egg develops into a morula. 5. The method of claim 1, wherein said embryo is human and said early developmental stage begins at the 4- to 8-cell stage. 6. The method of claim 1, wherein the supplementation of the medium is performed during the morula to early blastocyst developmental stages of the embryo. 7. The method of claim 1, wherein the supplementation of the medium is performed during the early developmental stage of the embryo to the morula stage and during the morula to early blastocyst development stage. 8. The method of claim 1, wherein said embryos are mice and said supplementation comprises exposing cultured embryos to a prostaglandin or the like between 24 and 42 hours after harvesting the 2-cell developmental stage embryos. 9. The method of claim 1, wherein said embryos are mice and said supplementation comprises exposing embryos to said prostaglandin or an analog thereof between 42 and 72 hours after harvesting the 2-cell developmental stage embryos. 10. A method for improving the implantation potential of in vitro fertilized embryos, comprising: The method according to claim 1 enhances the in vitro development of the embryo, thereby enhancing the hatching potential of the embryo and increasing the implantation potential in vivo. 11. The method of claim 9, comprising allowing said embryo to fully hatch, wherein fully hatching comprises releasing said embryo from the zona pellucida. 12. The method of claim 10, comprising implanting said embryo into a uterus of a mammalian host and correlating said complete hatching with establishment of a viable pregnancy. 13. A method for increasing the live birth potential of mammalian embryos fertilized in vitro, comprising: According to the method of claim 1, the in vitro development of the embryo is strengthened, so as to realize the enhanced hatching or the enhanced hatching potential of the embryo; introducing embryos with enhanced hatching potential into the uterus of a mammalian host; and The introduced embryos are implanted into said uterus, wherein the ability of the embryos to implant into the uterus of the host is enhanced relative to embryos implanted in the uterus of a similar host but without such enhancement. 14. A method for increasing the birth rate of mammalian embryo populations through in vitro fertilization, comprising: Strengthening the in vitro development of the embryo according to the method of claim 1, thereby realizing enhanced hatching or enhanced hatching potential of the embryo; introducing embryos with enhanced hatching potential into mammalian hosts; and The implanted embryos are grown in vivo such that the live birth rate with enhanced hatching or potential for enhanced hatching is greater than the live birth rate for embryos implanted without such enhancement. 15. An improved cell culture medium for early in vitro development of mammalian embryos, wherein the improvement comprises supplementing prostaglandins or analogues thereof in an amount effective to promote complete hatching of said embryos in vitro. 16. The medium of claim 15, wherein said amount of prostaglandin or analog thereof is effective to enhance the full hatching potential of said embryos after said embryos are removed from in vitro culture and transferred into the uterus of a mammalian host.

Images