Prostacyclin enhances the implantation and live birth potentials of mouse embryos

J.-C. Huang1,3, J.S. Goldsby1 and W.-S.A. Wun2

1 The University of Texas Health Science Center-Houston, Obstetrics and Gynecology-Division of Reproductive Endocrinology and 2 Obstetrical and Gynecological Associates, Houston, TX 77030, USA

3 To whom correspondence should be addressed at: The University of Texas Health Science Center-Houston, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, 6431 Fannin Street, MSB 3.604, Houston, TX 77030, USA. Email: jaou-chen.huang{at}uth.tmc.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Recently we reported that iloprost, a stable analogue of prostacyclin, enhanced mouse embryo hatching. Here we present a follow-up study to determine whether exposure to iloprost augments the implantation and live birth potentials of mouse embryos. METHODS: Two-cell embryos (C3B6F1) were harvested 42 h after HCG injection and cultured in medium supplemented with iloprost. After 48 h, the embryos were transferred to 2.5 day pseudopregnant gestational carriers. The number of gestation sacs was counted 72 h later; the number of live pups and the weight of pups and placentae were determined 14 days later. The implantation rate was defined as gestation sac per embryo transferred; the live birth rate was defined as live pup per embryo transferred. RESULTS: The prostacyclin analogue enhanced the implantation rate from 42 to 76% [relative risk 1.84, 95% confidence interval (CI) 1.38–2.43]. The rate of live pups also increased from 28 to 36% (relative risk 1.28, 95% CI 1.04–1.56). The weights of the pups and of the placentae of the two groups were comparable. CONCLUSION: Prostacyclin enhances the potentials of implantation and live birth of mouse embryos.

Key words: cyclooxygenase/embryotrophic factor/prostaglandin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Environments within the oviduct promote preimplantation embryo development. Human embryos co-cultured with homologous oviduct cells had higher hatching rates (Yeung et al., 1992Go). If transferred, these embryos yielded higher live birth rates and lower abortion rates than control embryos (Yeung et al., 1996Go). Mouse embryos co-cultured with human oviduct epithelial cells also had a higher hatching rate (Xu et al., 2000Go) and less cell death (Xu et al., 2001Go). These results were attributed to soluble factors produced by oviduct epithelial cells.

Improved human IVF outcome in the last decade is due in part to improved embryo culture media, which have been modified based on the composition of human oviduct fluid (Quinn, 2004Go) and the metabolic needs of developing embryos (Gardner, 1998Go). Growth factor has been proposed to function as a paracrine or an endocrine factor between the embryo and the female reproductive tract (Hardy and Spanos, 2002Go). Based on insulin-like growth factor-I (IGF-I) levels in the fluid of human oviduct and augmented development of cultured human embryos by IGF-I, supplementing embryo culture media with IGF-I has been proposed as a means to improve IVF outcome (Lighten et al., 1998Go).

Prostacyclin (PGI2) traditionally is considered a regulator of blood and vascular homeostasis. Observations on cyclooxygenase-2 (COX-2) knock-out mice suggest that PGI2 may have other physiological functions, such as endometrial decidualization (Lim et al., 1997Go, 1999Go). In 2001, we serendipitously discovered that human oviduct epithelial cells express enzymes crucial to PGI2 synthesis, i.e. COX-1, COX-2 and PGI2 synthase, and, when incubated with excess arachidonic acid, produced a large amount of PGI2 (Huang et al., 2002Go). Similar findings are found in mouse oviducts (unpublished data). Subsequent studies showed that mouse embryos express receptors for PGI2, and supplementing culture media with iloprost, a stable analogue of PGI2, enhanced mouse embryo hatching (Huang et al., 2003Go).

In an attempt to correlate the enhanced embryo hatching with increased implantation and live births, mouse embryos cultured in media supplemented with iloprost were transferred to gestational carriers. The number of gestation sacs and live pups was compared with that of control embryos. Because additives in culture media reportedly increased fetal weights (Thompson et al., 1995Go; Sinclair et al., 1999Go; DeBaun et al., 2002Go), the weights of the pups and the placentae were also compared. Our results indicate that iloprost enhanced the potentials of implantation and live birth of mouse embryos without affecting the weights of the pups or the placentae.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Source of chemicals and institutional approval
Unless stated otherwise, the chemicals were purchased from Sigma Co. (St Louis, MO). The research protocols were approved by the Animal Welfare Committee at the University of Texas Health Science Center at Houston.

Harvest and culture of mouse embryos
Mouse embryos were harvested and cultured as described previously (Huang et al., 2003Go). Briefly, 2-cell embryos (C3B6F1) were harvested from superovulated, 3-week old C57Bl/6 female mice 42 h after HCG injection. The embryos (14 per group) were cultured at 37°C under 5% CO2 in a 4-well dish (Nalge Nunc International, Naperville, IL) each containing 600 µl of HTF medium (SAGE Biopharma, Bedminster, NJ). The experimental embryos received iloprost (1 µM, Caymen Chemical, Ann Arbor, MI) in water and the control embryos received an equal amount of water. In either case, the volume of water was <0.1% of the culture media.

Embryo transfer
The day after the embryo donors received HCG, vaginal smears were obtained from potential gestational carriers (8-week-old C3B6F1 female mice) to determine the stage of the estrus cycle. Those in estrus were paired with vasectomized ICR males (Harlan) and checked for vaginal plugs on the following morning. Those with plugs were designated to be 0.5 day pseudopregnant.

Embryo transfer was performed on day 2.5 of pseudopregnancy under a dissecting microscope (Olympus SZ-PT). Surgical anaesthesia was obtained through i.p. injection of ketamine (200 mg/kg) and xylazine (10 mg/kg) (both from Burns Vet Supply Inc., TX). Each uterine horn was accessed via a 2 cm flank incision. With the proximal oviduct held by a pair of forceps, an opening was created at the distal end of the uterine horn on the anti-mesenteric side with a 30 gauge needle. The opening permits the entry of the transfer pipette which has an inner diameter of 135 µm (MidAtlantic Diagnostics, Inc., NJ). Up to seven embryos in 0.8 µl of transfer medium [{alpha}-minimal essential medium (MEM) with 25 mM HEPES and 1% bovine serum albumin (BSA)] were transferred to each horn. After each transfer, the contents of the pipette were examined under a stereomicroscope to identify retained embryos. The flank incision was closed with 9 mm metallic clips (Clay Adams, Parsippany, NJ). To avoid mixing of control and experimental embryos due to embryo migration from one horn to the other (Dr Andreas Zimmer, University of Bonn, Germany, personal communication), each gestational carrier received either control or experimental embryos. To maintain consistent transfer techniques, the embryo transfer was performed according to the same protocol and by one individual (J.-C.H.), who was blinded to the treatment embryos received. To ensure that both groups benefited equally from the experience gained over the course of the study (~6 months), the embryos were assigned treatments (control or iloprost) in blocks of four.

Determination of implantation rate
At 72 h after embryo transfer, implantation was determined based on a previously described method with some modifications (Paria et al., 1993Go). Briefly, 3 min before euthanasia, 0.1 ml of Chicago blue (1%) was injected via the tail vein of the gestational carrier. After the carrier was sacrificed, the uterine horns were removed. Discrete blue bands circling the uterine horns were counted (Figure 1A). These bands reflect the increased vascular supply to the implantation sites. The uterine horns were then opened and the gestation sacs counted (Figure 1B). The implantation rate was expressed as the number of gestation sacs per embryo transferred.



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Figure 1. Implantation was determined based on increased vascularity at the implantation site and confirmed by gestation sac. At 72 h after embryo transfer, the gestational carrier was given 1% Chicago blue via the tail vein and euthanized 3 min later. (A) Discrete blue bands (indicated by *) circled the uterine horn that received embryos. (B) Each blue band corresponded to a gestation sac, which appeared as pink-coloured tissue in the opened uterine horn. No blue bands or gestation sacs were found in the uterine horn that did not receive embryos (marked by an arrow).

 
Determination of live birth rate and the weights of pups and placentae
Preliminary studies showed that some gestational carriers cannibalize their pups when the litter size is small. In order to count and weigh the pups accurately, we sacrificed gestational carriers 14 days after embryo transfer (16.5 days of pregnancy or 2 days before natural birth). After euthanasia, the number of live pups was counted and the weight of individual pups and placentae was determined. All pups were examined for gross anomalies. The number of empty sacs was also counted in order to determine the implantation rate. The live birth rate was expressed as the number of live pups per embryo transferred; the implantation rate was expressed as the number of sacs, with or without live pups, per embryo transferred.

Statistical analysis
Fisher's exact test was used to compare the rates; the Student's t-test was used to compare the weights. A p<0.05 was considered as statistically significant. The GraphPad Instat® software (GraphPad Software Inc., San Diego, CA) was used for statistical analysis.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
PGI2 enhanced embryo implantation
To determine whether the PGI2 analogue enhanced the implantation potential of mouse embryos, we compared the number of gestation sacs 72 h after embryo transfer. Eighty-four control embryos and 81 iloprost-treated embryos were transferred to 12 gestational carriers (Table I). At the time of transfer, the developmental stages of the embryos were comparable (Table I). Seventy-two hours later, more gestation sacs were found in the iloprost-treated group than in the control group [76 versus 42%, relative risk 1.84, 95% confidence interval (CI) 1.39–2.43, P<0.0001] (Table I).


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Table I. Effects of prostacyclin on the implantation mouse embryos

 
PGI2 enhanced the potential of live birth
Once we determined that exposure to PGI2 analogue enhanced the implantation of mouse embryos, we went on to investigate whether the same treatment would increase the potential for live birth. To avoid the gestational carriers cannibalizing their pups and to determine the weights of pups and placentae accurately, we euthanized gestational carriers 14 days after transfer (pregnancy day 16.5, ~2 days prior to natural birth). Four hundred and six control embryos and 415 iloprost-treated embryos were transferred to 30 and 31 gestational carriers, respectively. The live birth rates of the control and the experimental groups were 28 and 36%, respectively (P=0.017, relative risk 1.28, 95% CI 1.044–1.560, Table II). The weights of the pups and the placentae were comparable (Table II). Thus, the PGI2 analogue enhanced the potential for live birth of mouse embryos without affecting the weights of pups or placentae. In addition, no gross anomalies were noted.


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Table II. Effects of prostacyclin on the live birth potential of mouse embryos

 
There were 73 and 97 empty sacs in the control and the experimental groups, respectively. The implantation rates of control and experimental embryos, therefore, were 46 and 59%, respectively (P=0.002, relative risk 1.28, CI 1.12–1.46). These data further validate the enhanced embryo implantation by iloprost based on the number of gestation sacs examined 72 h after embryo transfer.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this follow-up study, we showed that supplementing culture media with a PGI2 analogue enhanced the implantation and potential live births of mouse embryos. These results corroborate our previous observation that a PGI2 analogue enhanced complete hatching of mouse embryos in culture (Huang et al., 2003Go); they also support our speculation that oviduct-derived PGI2 may serve a physiological function (Huang et al., 2002Go).

Approximately 60% of experimental and control embryos were at the morula stage at the time of transfer (Table I), yet transferred experimental embryos yielded more gestation sacs. These results validate our previous observation that exposure to a PGI2 analogue between the 8-cell and morula stages was sufficient to enhance the complete hatching of mouse embryos (Huang et al., 2003Go). They are also consistent with oviduct-derived PGI2 serving as an embryotrophic factor, because fertilized mouse eggs develop into morulae inside the oviduct (Snell and Stevens, 1966Go).

The above phenomenon suggests that the PGI2 analogue initiated a chain of events culminating in enhanced hatching, implantation and increased live births. In this regard, PGI2 is reminiscent of platelet-activating factor (PAF): short-term exposure to PAF increased the implantation of mouse (O'Neill, 1998Go) and human embryos (O'Neill et al., 1989Go). The molecular and cellular events launched by the PGI2 analogue must be critical to hatching. They may include increased embryonic cell number (Montag et al., 2000Go), augmented production of trypsin-like proteases by the trophectoderm (Perona and Wassarman, 1986Go; Sawada et al., 1990Go), enhanced blastocoele expansion due to the enhanced Na+–K+-ATPase system of the trophectoderm (Biggers et al., 1988Go) or yet to be discovered mechanism(s).

The implantation rates of control embryos were comparable (42 and 46%) when examined 72 h or 14 days after transfer, respectively. On the other hand, the implantation rate of experimental embryos examined 14 days after transfer (59%) was significantly less (P=0.038) than that examined 72 h after transfer (76%). It is plausible that more sacs were completely resorbed in the experimental group due to crowding of implantation sites. Alternatively, iloprost ‘rescued’ some embryos that would otherwise not have implanted, and only a fraction of those ‘rescued’ embryos developed into live pups.

Extrapolating the results of this study to human IVF should be done with caution. The percentage of genetically abnormal embryos used in the current study was likely to be lower than that of embryos in human IVF, because the former were from sexually immature mice. In addition, the gestation of mouse and human is fundamentally different: mice are litter animals whereas humans are not. Nevertheless, results from this study and abundant PGI2 production by human (Huang et al., 2002Go) and mouse (unpublished observation) oviducts suggest that PGI2 may be one of the embryotrophic factors secreted by the oviducts and that supplementing embryo culture media with a PGI2 analogue may improve IVF outcome.

Supplementing IVF culture media with substances that enhance embryo development or implantation has been adopted by most laboratories and media manufacturers with great care. The implantation of human embryos reportedly was enhanced by brief exposure to PAF (O'Neill et al., 1989Go) or co-culture with human oviduct epithelia (Yeung et al., 1996Go). The development of human embryos was also enhanced by co-culture with human oviduct epithelia (Yeung et al., 1992Go) or supplementing the culture media with IGF-I ligand (Lighten et al., 1998Go). However, none of the above measures is used routinely in human IVF. The concerns may include issues regarding reproductive toxicology, teratogenecity and, possibly, epigenetic effects of such manipulation. Lambs from ovine embryos cultured in media supplemented with human serum were heavier and had longer gestation (Thompson et al., 1995Go; Holm et al., 1996Go; Sinclair et al., 1999Go). A congenital disorder involving overgrowth and neoplasia, the Beckwith–Wiedermann syndrome, was associated with assisted reproductive technology (DeBaun et al., 2002Go; Maher et al., 2003Go).

Reproductive toxicology studies on iloprost (Battenfeld et al., 1995Go) showed that embryonic and fetal development was not affected in rabbits and monkeys, but digit reduction was observed in rats. The digit anomaly is likely to be due to reduced uteroplacental flow causing hypoxia of the affected structure rather than the inherent teratogenicity of iloprost, because iloprost is a vasodilator and the studied animals received iloprost throughout the gestation period. A similar defect could be induced by vasodilators with different chemical structures, such as hydralazine and dihydropyridines. In the current study, the exposure to iloprost took place during the preimplantation period. Among the 246 live pups examined, there was no digit reduction or other anomalies, and the weights of pups and placentae between the two groups were similar. Despite all these, supplementing IVF media with a PGI2 analogue should be considered as an experimental protocol.

In summary, we showed that a PGI2 analogue enhanced the implantation and live birth potentials of mouse embryos without affecting the weights of pups and placentae. However, until follow-up studies on mice exposed to iloprost during the preimplantation period become available, PGI2 analogues should not be used in human IVF.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors wish to thank Ms Dana Whittaker for her secretarial assistance. J.-C.H. is a Women's Reproductive Health Research Scholar (NICHD HD01277).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 16, 2004; accepted on May 11, 2004.