Reproductive Sciences http://rsx.sagepub.com/

Embryo Transfer Catheter Contamination With Intravaginal Progesterone Preparations in a Simulated Embryo Transfer Model Impairs Mouse Embryo Development: Are There Implications for Human Embryo Transfer Technique? Luke Y. Ying, Ying Ying, James Mayer, Anthony N. Imudia and Shayne M. Plosker Reproductive Sciences published online 10 February 2014 DOI: 10.1177/1933719114522522 The online version of this article can be found at: http://rsx.sagepub.com/content/early/2014/02/06/1933719114522522

Published by: http://www.sagepublications.com

On behalf of:

Society for Gynecologic Investigation

Additional services and information for Reproductive Sciences can be found at: Email Alerts: http://rsx.sagepub.com/cgi/alerts Subscriptions: http://rsx.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav

>> OnlineFirst Version of Record - Feb 10, 2014 What is This?

Downloaded from rsx.sagepub.com at Scientific library of Moscow State University on February 17, 2014

Original Article

Embryo Transfer Catheter Contamination With Intravaginal Progesterone Preparations in a Simulated Embryo Transfer Model Impairs Mouse Embryo Development: Are There Implications for Human Embryo Transfer Technique?

Reproductive Sciences 1-6 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719114522522 rs.sagepub.com

Luke Y. Ying, BS1, Ying Ying, PhD1, James Mayer, MD1, Anthony N. Imudia, MD1, and Shayne M. Plosker, MD1

Abstract Objectives: To study the effect of embryo transfer (ET) catheter contact with intravaginal progesterone preparations on mouse embryo development. Study Design: In a simulated ET model, ET catheters were loaded with culture medium, placed in contact with intravaginal progesterone gel (Crinone 8%) or micronized progesterone intravaginal inserts (Endometrin 100 mg), and the intracatheter culture medium flushed. Embryos were cultured in the flushed culture medium at variable dilutions for variable lengths of time. Proportion of embryos progressing to blastocyst, embryo cell number, and apoptotic index was analyzed. Results: None of the embryos cultured in undiluted progesterone-exposed medium progressed to blastocyst. The likelihood of achieving blastocyst status and the average embryo cell number increased significantly as culture media exposed to intravaginal progesterone was diluted. A significant decrease in cell number became apparent between 1 and 2 hours of exposure. Interestingly, the apoptotic index was significantly higher in progesterone-exposed embryos as compared to unexposed embryos. Conclusion: The contamination of ET catheter with intravaginal progesterone significantly impairs mouse embryo development, likely due in part to increased programmed cell death. Keywords Crinone, Endometrin, embryo toxicity, embryo transfer, progesterone

Introduction Progesterone, produced initially in the ovarian corpus luteum and later in the placenta during pregnancy, is essential for successful implantation and for maintaining a viable ongoing pregnancy.1,2 Early studies from human oocyte donation3,4 and frozen embryo transfer (ET) cycles5 demonstrated the necessity of progesterone, and the narrow implantation window, for ET after progesterone initiation, in order to achieve pregnancy. In addition to inducing endometrial structural changes that favor implantation,6 the role of progesterone in the luteal phase of the menstrual cycle and early pregnancy may include modulation of fallopian tube peristalsis to facilitate the transport of the zygote to the endometrial cavity7 and myometrial relaxation to favor implantation.8 As gonadotropin-releasing hormone analogs are administered to the majority of women undergoing in vitro fertilization (IVF), progesterone supplementation is necessary in order to achieve optimal pregnancy rates.9 However, the optimal duration of exogenous progesterone supplementation10 and the

optimal route of progesterone administration have been under contentious debate. A Cochrane meta-analysis concluded that ongoing pregnancy rates and live birth rates were higher with intramuscular progesterone compared with intravaginal progesterone.11 However, more recent studies have resulted in conflicting conclusions12-15 even considering 2 independent prospective trials conducted in the same institution.13,14 There is currently no biological plausibility that either intravaginal or intramuscular routes of progesterone administration would be superior to one another. Serum progesterone levels similar to

1 Department of Obstetrics and Gynecology, USF IVF and Reproductive Endocrinology, University of South Florida Morsani College of Medicine, Tampa, FL, USA

Corresponding Author: Shayne M. Plosker, Department of Obstetrics and Gynecology, University of South Florida, 2 Tampa General Circle, 6th Floor (6025), Tampa, FL 33606, USA. Email: [email protected]

Downloaded from rsx.sagepub.com at Scientific library of Moscow State University on February 17, 2014

2

Reproductive Sciences

those observed physiologically in the luteal phase of nonmedicated natural cycles are observed after daily intramuscular injections of 25 mg of progesterone16 and intravaginal insertions of 100 mg of progesterone twice or 3 times daily.17 Histological studies of the endometrium demonstrate in-phase secretory conversion of proliferative endometrium in response to intravaginal progesterone administration, even when serum progesterone values are less than 5 ng/mL.18 During IVF cycles, it is routine to initiate progesterone supplementation 24 to 48 hours postoocyte retrieval. In patients utilizing intravaginal preparations, intravaginal progesterone particles that are adherent to the vaginal wall or cervix are removed through rigorous and gentle washing with saline and culture medium at the time of ET, which in some cases might be a very difficult task to completely achieve. The difference in clinical practice style as it relates to cervical preparation during ET might provide explanation to the discrepant results reported in the literature regarding the efficacy of intramuscular versus intravaginal progesterone supplementation in IVF. To this end, we hypothesized that the possible contamination of ET catheter tip with intravaginal progesterone particles at the time of ET might have a detrimental effect on embryo development. Mouse embryo toxicity bioassays are a gold standard in quality control testing in the IVF laboratory, used to evaluate culture media, culture media supplements, raw materials, and various disposable items such as culture dishes. We sought to study the effects of intravaginal progesterone on embryo development, using mouse embryo toxicity bioassays, by developing a model that would simulate the effects of exposure of human embryos to intravaginal progesterone at the time of ET.

of the catheters were then dipped in progesterone gel (PG, Crinone 8%; Columbia Laboratories, Livingston, New Jersey) or micronized progesterone (MP) for few seconds. The MP was prepared by adding 1 tablet of Endometrin 100 mg (Ferring Pharmaceuticals Inc, Parsippany, New Jersey) to 1 mL of saline and keeping in a 37 C incubator for 1 hour. The media within the ET catheters were flushed out through the tip of the PG- or MP-exposed catheter tip by pushing the 1-mL syringe. The PG- or MP-exposed media was gently mixed and aliquoted in 10 mL drops in a 60-mm Falcon dish and covered with Quinn Advantage Mineral Oil (Cooper Surgical Inc) or diluted with 10% SPS Quinn Advantage Cleavage Medium before being aliquoted. The 10% SPS Quinn Advantage Cleavage Medium was used as a progesterone-absent control.

Dose–Response Study The thawed mouse embryos were rinsed through 3 drops of preequilibrated control media before being placed in culture with 5 embryos in each drop. Embryos were cultured in undiluted PG- (n ¼ 27) or MP-flushed media (n ¼ 23) as well as media at 1:2 dilutions for PG (n ¼ 32) and MP (n ¼ 27) and 1:4 dilutions for PG (n ¼ 40) and MP (n ¼ 26). For control, 10% SPS Quinn Advantage Cleavage Medium was used (n ¼ 74). After 48 hours of culture, the proportion of embryos that developed into blastocyst was determined and the number of blastomeres was counted after fixation.

Time Course Study Materials and Methods Mouse Embryo Preparation Commercially available mouse embryos from B6C3F-1  B6D2F-1 breeding cryopreserved at the 2-cell stage (Conception Technology, San Diego, California) were thawed according to the instructions. Straws with embryos were kept at room temperature for 2 minutes and the embryos were then washed in 10% serum protein substitute (SPS)-modified HTF (Cooper Surgical Inc, Trumbull, Connecticut), and this condition and process were the same for all the embryos utilized for all experiments. After several washes, the embryos were exposed to progesterone preparations at various dilutions (dose–response experiments) and for variable durations of time (time course experiments).

Embryo Exposure to Intravaginal Progesterone Preparations To simulate human ET, ET catheters (EchoTip Soft-Pass Embryo Transfer Catheter; Cook Medical Inc, Bloomington, Indiana) attached to a 1-mL syringe were prepared by washing 3 times before they were loaded with 30 mL of 10% SPS Quinn Advantage Cleavage Medium (Cooper Surgical Inc). The tips 2

Embryos were cultured in undiluted flushed PG-exposed medium for 1 hour (n ¼ 26) or 2 hours (n ¼ 26) and then transferred into 10% SPS Sage Cleavage Medium for further culture. For control, 10% SPS Quinn Advantage Cleavage Medium was used (n ¼ 28). Assessment of embryo development (blastocyst formation) was performed at 48 and 72 hours of culture. The number of blastomeres was counted at 72 hours after fixation.

Blastocyst Fixation and Blastomere Count After 48 or 72 hours of culture, blastocysts were suspended in 100 mL of hypotonic solution of 0.7% sodium citrate for 10 minutes. Then, the blastocysts were placed on Fisherbrand Superfrost microscope slides (ThermoFisher Scientific, Waltham, Massachusetts) in the minimum amount of hypotonic solution. A small drop of fixative of methanol and acetic acid (2:1) was added directly onto the top of the blastocysts. When the hypotonic solution around the blastomeres had completely disappeared, more drops of fixative were added. After drying for 20 minutes, the slides were stained with modified 2% Giemsa for 10 minutes and the number of blastomeres was counted under a phase-contrast microscope (400 magnification).

Downloaded from rsx.sagepub.com at Scientific library of Moscow State University on February 17, 2014

Ying et al

3

Measurement of Apoptosis in Embryos DNA fragmentation by apoptosis was measured by use of the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay using the In Situ Cell Death Detection Kit (Roche Diagnostics Corporation, Indianapolis, Indiana). Embryos were exposed in undiluted PG-flushed medium for 2 hours prior to transferring into 10% SPS Sage Cleavage Medium for total of 72 hours (n ¼ 23). For control, embryos were cultured in 10% SPS Quinn Advantage Cleavage Medium for 72 hours (n ¼ 21). The embryos were washed in phosphate-buffered saline (PBS) containing 0.1% polyvinylpyrrolidone (PVP) and then fixed in 3.7% paraformaldehyde in PBS for 1 hour at room temperature. The embryos were then washed 3 times in PBS/PVP and permeabilized in 0.5% Triton X-100 for 1 hour at room temperature. After being washed in PBS/PVP, the embryos were incubated at 37 C for 1 hour in darkness in the TUNEL reaction solution and counterstained with 50 mg/mL propidium iodide (Molecular Probes; Sigma, St Louis, Missouri). The embryos were then washed thoroughly and mounted on Fisherbrand Superfrost microscope slides with 40 ,6diamidino-2-phenylindole hydrochloride antibleaching solution. The slides were sealed with clear nail varnish and stored in the dark at 20 C until analysis by fluorescent microscope. The TUNEL-positive (apoptotic) nuclei appeared green and the normal chromatin appeared blue and red.

Table 1. Dose Effects of PG and MP on Mouse Embryo Development After 48 Hours of Culture.a Treatment Control PG 1:0 PG 1:2 PG 1:4 MP 1:0 MP 1:2 MP 1:4

No. of Embryos

% of Blastocysts

74 27 32 40 23 27 26

90.5 0 40.6b 65.0d 0 44.4b 53.8b

Number of Blastomeres, Mean + SEM (n) 34.7 + 4.8 + 23.6 + 32.1 + 4.8 + 23.6 + 25.5 +

1.3 (42) 0.4 (25)b 1.8 (24)c 1.9 (29)e 1.4 (20)b 1.9 (24)c 2.0 (23)f

Abbreviations: MP, micronized progesterone; n, the number of embryos that were fixed; PG, progesterone gel; SEM, standard error of the mean. a All compared with the controls. b P < .0001. c P < .001. d P < .01. e P > .05. f P < 0.05.

+ 1.3; Figure 1 and Table 1). At 1:4 dilution, the mean + SEM blastomeres in the PG group were not significantly different from that of the control (32.1 + 1.9 vs 34.7 + 1.3, P > .05); however, the number of blastomeres in MP was significantly less than that of the control (25.5 + 2.0 vs 34.7 + 1.3, P < .05). The proportion of embryos that developed into blastocyst and the number of blastomeres between the 2 different progesterone preparation groups were not significantly different from each other (Table 1).

Statistical Analysis Differences in blastocyst formation between the different treatment groups were analyzed using the chi-square test. The number of blastomeres and the apoptotic index were expressed in mean + standard error of the mean (SEM). Differences were analyzed using analysis of variance or Student t test as appropriate. P < .05 was considered statistically significant.

Results Dose Effects of PG and MP on Mouse Embryo Development None of the embryos cultured with undiluted PG- or MP-exposed flushed medium developed to blastocyst stage (Table 1). To further evaluate whether the putative inhibition of embryo development by PG and MP was dose dependent, the embryos that were cultured in 1:2 and 1:4 diluted PG- or MP-exposed flushed media were reviewed. At 1:2 dilution, 40.6% of embryos in PG and 44.4% in MP developed to blastocyst; these blastulation rates were significantly lower than the 90.5% seen in the control group (P < .0001). At 1:4 dilution, 65.0% of embryos in PG and 53.8% in MP developed to blastocyst which were also significantly lower than that of the control group (P < .01 and P < .0001, respectively, Table 1). In the 1:2 dilution groups, the mean + SEM blastomere numbers were 23.6 + 1.8 (P < .001) and 23.6 + 1.9 (P < .001) for the PG and MP, respectively, when compared with controls (34.7

Time Course Effects of PG on Mouse Embryo Development As previously shown, none of the mouse embryos developed to blastocyst stage when cultured in undiluted PG-flushed medium for 48 hours. To elucidate how long the embryos needed to be exposed to undiluted PG-flushed medium before their development was compromised, we further studied time course effects of PG on mouse embryo development. After 2 hours of exposure to undiluted PG-flushed medium, 61.5% of embryos developed to blastocyst stage, which was significantly lower than 92.9% in the control (P < .05). The mean number of blastomeres was 43.8 + 4.5 compared to 60.2 + 3.4 in the control (P < .05, Table 2). However, after 1-hour exposure to undiluted PG-flushed medium, there was no significant difference in terms of percentage of blastocyst formation and average number of blastomeres when compared to control (Table 2).

Apoptotic Effects of PG on Mouse Embryo Development Given the clear correlation between the dose and the length of exposure of progesterone to mouse embryo developmental potential, we evaluated the apoptotic index of the exposed and control embryos as a way to further understand possible mechanisms of PG-induced impairment of mouse embryo development. The embryos that were exposed for 2 hours in undiluted PG-flushed medium and cultured for additional 70

Downloaded from rsx.sagepub.com at Scientific library of Moscow State University on February 17, 2014

3

4

Reproductive Sciences

Figure 1. Representative micrographs of blastocysts after culture in the 10% serum protein substitute (SPS) Quinn Advantage Cleavage Medium (A) and blastomeres after the fixation (B), 1:2 diluted progesterone gel medium (C), and blastomeres after fixation (D).

Table 2. Time Course Effects of PG on Mouse Embryo Development After Exposure With Contaminated Medium.a Treatment

No. of Embryos

% of Blastocysts

Number of Blastomeres, Mean + SEM (n)

Control PG 1 hour PG 2 hours

28 26 26

92.9 88.5b 61.5c

60.2 + 3.4 (26) 51.9 + 4.5 (19)b 43.8 + 4.7 (20)c

Abbreviations: n, the number of embryos that were fixed; PG, progesterone gel; SEM, standard error of the mean. a All compared with the controls. n: the number of embryos that were fixed. b P > 0.05. c P < 0.05.

hours in control media showed almost 2-fold higher apoptotic index on TUNEL analysis when compared to unexposed embryos (14.6% vs 7.9%, P < .01; Figure 2).

Discussion In this study, dose- and time-dependent mouse embryotoxicity was demonstrated when mouse embryos were cultured in media exposed to 2 intravaginal progesterone preparations (PG and 4

MP) in current clinical use. No embryo developed to the blastocyst stage after cultured in undiluted PG- or MP-exposed media. With serial dilutions of progesterone-exposed media, the likelihood of blastocyst development and the mean blastomere number, increased. The embryotoxic effect was demonstrated between 1 and 2 hours of culture in PG-exposed media. The finding of higher apoptotic index in the PG-exposed embryos suggests that the putative mechanism for direct embryotoxicity is mediated in part by increased programmed cell death (apoptosis), although the possibility of inhibition of cell division could also be operational. Biologically, progesterone plays a fundamental role in embryo implantation1,2 mainly because of the different organspecific cellular and structural changes that favor implantation.6 Such changes include modulation of fallopian tube peristalsis to facilitate transport of the zygote to the endometrial cavity,7 myometrial relaxation8 and induction of endometrial expression, and secretion of different growth factors and cytokines necessary to enhance endometrial receptivity and implantation.19-21 Although these are indirect effects of progesterone on embryo development as evidenced by improved implantation, no study has evaluated the direct impact of different preparations of progesterone on the in vitro embryo-developmental potential under normal laboratory culture conditions. Specifically, exposure of

Downloaded from rsx.sagepub.com at Scientific library of Moscow State University on February 17, 2014

Ying et al

5

18

n = 23

16

Apoptotic Index (%)

14 12 10

n = 21

8

14.6

6 4

7.9

2 0

Control

PG 2h

Figure 2. Apoptotic index of embryos cultured in the progesterone gel (PG)-contaminated medium for 2 hours compared with control (P < .01). The numbers on top of the bars represent the number of embryos cultured.

mouse embryos to progesterone results in significant impairment of embryo development (blastulation rates and number of blastomeres). Under other conditions, progesterone has been found to have different effects on early embryo development. For example, complement-mediated chick embryotoxicity of human sera was attenuated as progesterone levels increase.22 Progesterone was shown to inhibit in vitro embryotoxic T-helper 1 cell cytokine production to trophoblast in women with recurrent pregnancy loss.23 It is also important to note that Harini et al reported that administration of progesterone induces preimplantation embryonic loss in mice.24 This effect was observed after administration of supraphysiologic dose of progesterone in the peri-implantation period, suggesting that progesterone may have detrimental effect on mouse embryo development and implantation. Although the observed impairment in embryo development is being attributed directly to progesterone, it is possible to speculate that other components of the preparations could play a role. Progesterone gel is a preparation of MP in oil and water emulsion composed of a water swellable, but insoluble polymer, polycarbophil. Micronized progesterone is contained in a base containing lactose monohydrate, PVP, adipic acid, sodium bicarbonate, sodium lauryl sulfate, magnesium stearate, pregelatinized starch, and colloidal silicon dioxide. Direct embryo contact to either intravaginal preparation could conceivably lead to alterations in the microenvironment surrounding the embryo, such as pH, solubility, and fluid balance alteration, or dilution of essential elements in culture medium or in the endometrial cavity. Since embryotoxic effects were noted between 1 and 2 hours of exposure, these effects could take place before the embryo has an opportunity to equilibrate in the endometrial cavity after ET.

This model developed to study the potential effects of intravaginal progesterone on embryo development reflects many, but not all, aspects of actual human ET. Similarities of the model to real ET were that the ET catheter was prepared in a similar fashion, and culture was undertaken in standard cleavage-stage culture medium flushed through the ET catheter, as would occur during actual ET. The ET catheter tips dipped in PG or MP are analogous to an ET catheter tip having inadvertent contact with an intravaginal PG or MP suspension/particle during ET. Different from true ET, the embryos were not loaded into the transfer catheter and were not transferred into the uterus but rather were cultured in vitro with the intravaginal progesterone-contaminated medium to study embryo development. Moreover, the extent to which mouse embryo observations can be extrapolated to human embryo development is uncertain. Notwithstanding, mouse embryotoxicity bioassays are a gold standard in quality control testing and troubleshooting in the IVF laboratory and used to evaluate culture media, culture media supplements, culture conditions, raw materials, and various disposable items such as culture dishes. As such, we believe that our observations are meaningful in and of themselves and may have potential clinical relevance to human IVF. Over the past decade, there has been debate about the optimal route of luteal phase progesterone supplementation during IVF. Two meta-analyses arrived at different conclusions11,12 and a group of investigators came to 2 different conclusions with 2 separate studies several years apart.13,14 Compelling evidence for better outcome of 1 route of administration versus the other is lacking.16-18 Paralleling the shift from older studies favoring intramuscular progesterone over intravaginal progesterone to more recent data showing equivalence in outcome between the 2 progesterone delivery routes, reproductive endocrinologists have placed greater emphasis on ET technique. The importance of rigorous and careful cleaning of the vagina and cervix, as well as irrigation of the endocervical canal at the time of ET, has been discussed. This is because of concern about deleterious effects of microbial contamination of the ET catheter tip25-27 and concern that the presence of cervical mucus could result in embryo expulsion. Older data suggesting that intravaginal progesterone was inferior may have been confounded by intravaginal progesterone-induced embryotoxicity during an era when less attention was paid to ET technique including rigorous and careful vaginal and endocervical preparation at the time of ET. In summary, we found that intravaginal progesterone preparations in current clinical use were embryotoxic to mouse embryos in a dose- and time-dependent fashion in a simulated ET model. Our data suggest that apoptosis plays a role. Avoiding contamination of the ET catheter tip with intravaginal progesterone to avoid embryotoxicity may be a previously unrecognized benefit of rigorous vaginal and cervical cleaning at the time of ET. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Downloaded from rsx.sagepub.com at Scientific library of Moscow State University on February 17, 2014

5

6

Reproductive Sciences

Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.

15.

References 1. Devoto L, Fuentes A, Kohen P, Cespedes P, et al. The human corpus luteum: life cycle and function in natural cycles. Fertil Steril. 2009;92(3):1067-1079. 2. Csapo AI, Pulkkinen MO, Wiest WG. Effects of luteectomy and progesterone replacement therapy in early pregnant patients. Am J Obstet Gynecol. 1973;115(6):759-765. 3. Navot D, Bergh PA, Williams M, et al. An insight into early reproductive processes through the in vivo model of ovum donation. J Clin Endocrinol Metab. 1991;72(2):408-414. 4. Rosenwaks Z. Donor eggs: their application in modern reproductive technologies. Fertil Steril. 1987;47(6):895-909. 5. Lelaidier C, de Ziegler D, Freitas S, Olivennes F, Hazout A, Frydman R. Endometrium preparation with exogenous estradiol and progesterone for the transfer of cryopreserved blastocysts. Fertil Steril. 1995;63(4):919-921. 6. Acosta AA, Elberger L, Borghi M, et al. Endometrial dating and determination of the window of implantation in healthy fertile women. Fertil Steril. 2000;73(4):788-798. 7. Wanggren K, Stavreus-Evers A, Olsson C, Andersson E, Gemzell-Danielsson K. Regulation of muscular contractions in the human Fallopian tube through prostaglandins and progestagens. Hum Reprod. 2008;23(10):2359-2368. 8. Bulletti C, de Ziegler D. Uterine contractility and embryo implantation. Curr Opin Obstet Gynecol. 2006;18(4):473-484. 9. Nosarka S, Kruger T, Siebert I, Grove D. Luteal phase support in in vitro fertilization: meta-analysis of randomized trials. Gynecol Obstet Invest. 2005;60(2):67-74. 10. Nyboe Andersen A, Popovic-Todorovic B, Schmidt KT, et al. Progesterone supplementation during early gestations after IVF or ICSI has no effect on the delivery rates: a randomized controlled trial. Hum Reprod. 2002;17(2):357-361. 11. Daya S, Gunby J. Luteal phase support in assisted reproduction cycles. Cochrane Database Syst Rev. 2004;(3):CD004830. 12. Zarutskie PW, Phillips JA. A meta-analysis of the route of administration of luteal phase support in assisted reproductive technology: vaginal versus intramuscular progesterone. Fertil Steril. 2009;92(1):163-169. 13. Yanushpolsky E, Hurwitz S, Greenberg L, Racowsky C, Hornstein M. Crinone vaginal gel is equally effective and better tolerated than intramuscular progesterone for luteal phase support in in vitro fertilization–embryo transfer cycles: a prospective randomized study. Fertil Steril. 2010;94(7):2596-2599. 14. Propst AM, Hill JA, Ginsburg ES, Hurwitz S, Politch J, Yanushpolsky EH. A randomized study comparing Crinone 8% and

6

16.

17.

18.

19.

20.

21.

22.

23.

24.

25. 26.

27.

intramuscular progesterone supplementation in in vitro fertilization–embryo transfer cycles. Fertil Steril. 2001;76(6):1144-1149. Silverberg K, Vaughn TC, Hansard L, Burger NZ, Minter T. Progesterone vaginal gel vs. intramuscular progesterone in oil for luteal support in IVF: a large, prospective trial. Fertil Steril. 2012;97(2):344-348. Tavaniotou A, Smitz J, Bourgain C, Devroey P. Comparison between different routes of progesterone administration as luteal phase support in infertility treatments. Hum Reprod Update. 2000;6(2):139-148. Blake EJ, Norris PM, Dorfman SF, Longstreth J, Yankov VI. Single and multidose pharmacokinetic study of a vaginal micronized progesterone insert (Endometrin) compared with vaginal gel in healthy reproductive-aged female subjects. Fertil Steril. 2010; 94(4):1296-1301. Fanchin R, De Ziegler D, Bergeron C, Righini C, Torrisi C, Frydman R. Transvaginal administration of progesterone. Obstet Gynecol. 1997;90(3):396-401. Liang X, Zhang XH, Han BC, et al. Progesterone and heparinbinding epidermal growth factor-like growth factor regulate the expression of tight junction protein Claudin-3 during early pregnancy. Fertil Steril. 2013;100(5):1410-1418. doi:10.1016/j.fertnstert.2013.07.001. Liu N, Zhou C, Chen Y, Zhao J. The involvement of osteopontin and b3 integrin in implantation and endometrial receptivity in an early mouse pregnancy model. Eur J Obstet Gynecol Reprod Biol. 2013;170(1):171-176. Herrler A, von Rango U, Beier HM. Embryo–maternal signalling: how the embryo starts talking to its mother to accomplish implantation. Reprod Biomed Online. 2003;6(2):244-256. Zeman M, Novakova P. Gestational progesterone suppresses embryotoxic action of the complement system to chick embryo. Neuro Endocrinol Lett. 2001;22(1):33-37. Choi BC, Polgar K, Xiao L, Hill JA. Progesterone inhibits in-vitro embryotoxic Th1 cytokine production to trophoblast in women with recurrent pregnancy loss. Hum Reprod. 2000;15 suppl 1:46-59. Harini C, Sainath SB, Reddy PS. Progesterone administration induces preimplantation embryonic loss in mice. Fertil Steril. 2009;91(5 suppl):2137-2141. Mains L, Van Voorhis BJ. Optimizing the technique of embryo transfer. Fertil Steril. 2010;94(3):785-790. Moore DE, Soules MR, Klein NA, Fujimoto VY, Agnew KJ, Eschenbach DA. Bacteria in the transfer catheter tip influence the live-birth rate after in vitro fertilization. Fertil Steril. 2000;74(6): 1118-1124. Egbase PE, Al-Sharhan M, al-Othman S, al-Mutawa M, Udo EE, Grudzinskas JG. Incidence of microbial growth from the tip of the embryo transfer catheter after embryo transfer in relation to clinical pregnancy rate following in-vitro fertilization and embryo transfer. Hum Reprod. 1996;11(8):1687-1689.

Downloaded from rsx.sagepub.com at Scientific library of Moscow State University on February 17, 2014

Embryo Transfer Catheter Contamination With Intravaginal Progesterone Preparations in a Simulated Embryo Transfer Model Impairs Mouse Embryo Development: Are There Implications for Human Embryo Transfer Technique?

To study the effect of embryo transfer (ET) catheter contact with intravaginal progesterone preparations on mouse embryo development...
258KB Sizes 0 Downloads 2 Views