Intrauterine human chorionic gonadotropin infusion in oocyte donors promotes endometrial synchrony and induction of early decidual markers for stromal survival: a randomized clinical trial

Abstract

Study question: Does a single intrauterine infusion of human chorionic gonadotropin (hCG) at the time corresponding to a Day 3 embryo transfer in oocyte donors induce favorable molecular changes in the endometrium for embryo implantation?

Summary answer: Intrauterine hCG was associated with endometrial synchronization between endometrial glands and stroma following ovarian stimulation and the induction of early decidual markers associated with stromal cell survival.

What is known already: The clinical potential for increasing IVF success rates using an intrauterine hCG infusion prior to embryo transfer remains unclear based on previously reported positive and non-significant findings. However, infusion of CG in the non-human primate increases the expression of pro-survival early decidual markers important for endometrial receptivity, including α-smooth muscle actin (α-SMA) and NOTCH1.

Study design, size, duration: Oocyte donors (n=15) were randomly assigned to receive an intrauterine infusion of 500 IU hCG (n=7) or embryo culture media vehicle (n=8) 3 days following oocyte retrieval during their donor stimulation cycle. Endometrial biopsies were performed 2 days later, followed by either RNA isolation or tissue fixation in formalin and paraffin embedding.

Participants/materials, setting, methods: Reverse transcription of total RNA from endometrial biopsies generated cDNA, which was used for analysis in the endometrial receptivity array (ERA; n = 5/group) or quantitative RT-PCR to determine relative expression of ESR1, PGR, C3 and NOTCH1. Tissue sections were stained with hematoxylin and eosin followed by blinded staging analysis for dating of endometrial glands and stroma. Immunostaining for ESR1, PGR, α-SMA, C3 and NOTCH1 was performed to determine their tissue localization.

Main results and the role of chance: Intrauterine hCG infusion was associated with endometrial synchrony and reprograming of stromal development following ovarian stimulation. ESR1 and PGR were significantly elevated in the endometrium of hCG-treated patients, consistent with earlier staging. The ERA did not predict an overall positive impact of intrauterine hCG on endometrial receptivity. However, ACTA2, encoding α-SMA was significantly increased in response to intrauterine hCG. Similar to the hCG-treated non-human primate, sub-epithelial and peri-vascular α-SMA expression was induced in women following hCG infusion. Other known targets of hCG in the baboon were also found to be increased, including C3 and NOTCH1, which have known roles in endometrial receptivity.

Limitations, reasons for caution: This study differs from our previous work in the hCG-treated non-human primate along with clinical studies in infertile patients. Specifically, we performed a single intrauterine infusion in oocyte donors instead of either continuous hCG via an osmotic mini-pump in the baboon or infusion followed by blastocyst-derived hCG in infertile women undergoing embryo transfer. Therefore, the full impact of intrauterine hCG in promoting endometrial receptivity may not have been evident.

Wider implications of the findings: Our findings suggest a potential clinical benefit for intrauterine hCG prior to embryo transfer on Day 3 in counteracting endometrial dyssynchrony from ovarian stimulation and promoting expression of markers important for stromal survival. Finally, there were no obvious negative effects of intrauterine hCG treatment.

Study funding/competing interests: Funding for this work was provided by NICHD R01 HD042280 (A.T.F.) and NICHD F30 HD082951 (M.R.S.). C.S. and P.D.-G are co-inventors of the patented ERA, which is owned by IGENOMIX SL and was used in this study, and C.S. is a shareholder in IGENOMIX SL. M.R.-A. is employed by IGENOMIX SL. No other authors have any conflicts of interest to report.

Trial registration number: This study was registered with ClinicalTrials.gov (NCT01786252).

Trial registration date: 5 February 2013.

Date of first patient's enrollment: 10 May 2013.

Keywords: Notch; complement; controlled ovarian stimulation; decidualization; embryo implantation; endometrial receptivity; endometrium; hCG; oocyte donors.

© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Figures

Figure 1

Expression of steroid hormone receptors, ESR1 and PGR, detected by quantitative RT–PCR and immunohistochemistry after intrauterine infusion of hCG or vehicle. (A) Both ESR1 and PGR mRNA expression were significantly increased in hCG-treated patients. (B) Consistent with mRNA expression, both endometrial glandular epithelial and stromal compartments from hCG-treated patients expressed significantly elevated protein levels of both ESR1 and PGR. Staining intensity for glandular epithelial (GE) and stromal compartments was quantified by image analysis software ImageJ (D-HSCORE). *P < 0.05, **P < 0.01; Scale bar = 100 µm.

Figure 2

Validation of endometrial hCG effect by the induction of epithelial ERK1/2 phosphorylation. Immunostaining for ERK1/2 and phospho-ERK1/2 was performed in oocyte donors after intrauterine infusion of vehicle or hCG. Intrauterine hCG resulted in significantly elevated levels of ERK1/2 in both endometrial glandular epithelium (GE) and stroma. Phosphorylation of ERK1/2 was significantly increased in GE in response to hCG but not in the stroma. Staining intensity for GE and stromal compartments was quantified by image analysis software ImageJ (D-HSCORE) for statistical analysis. *P < 0.05, **P < 0.01; Scale bar = 100 µm.

Figure 3

Immunolocalization of endometrial α-SMA in response to intrauterine hCG infusion in oocyte donors. Intrauterine hCG resulted in significantly increased stromal expression of α-SMA compared with vehicle controls. Similar to the non-human primate, α-SMA expression localized primarily to the sub-luminal epithelial (arrow) and peri-vascular stromal cells. Staining intensity was quantified by image analysis software ImageJ (D-HSCORE) for statistical analysis. **P < 0.01; Scale bar = 50 µm.

Figure 4

Expression of NOTCH1 and C3 in the endometrium of oocyte donors following intrauterine hCG infusion. (A) Intrauterine hCG did not significantly impact mRNA expression levels of NOTCH1 or C3 by quantitative RT–PCR. (B) Both endometrial glandular epithelial (GE) and stromal compartments from hCG-treated patients expressed significantly elevated levels of NOTCH1 protein. Consistent with our findings in the non-human primate, intrauterine infusion of hCG significantly increased endometrial stromal C3 protein expression while there was no significant impact on C3 expression in the GE. Staining intensity for GE and stromal compartments was quantified by image analysis software ImageJ (D-HSCORE) for statistical analysis. *P < 0.05, **P < 0.01; Scale bar = 100 µm.

Figure 5

Working hypothesis of mechanisms by which intrauterine hCG regulates key signaling mechanisms in the endometrial stroma. PGR coordinates hCG-induced responses and may in itself be regulated by hCG based on the current study. C3 acts downstream of PGR to modulate the endometrial immune response for implantation. NOTCH1 can directly act to promote stromal survival for decidualization or regulate α-SMA, which is necessary for both stromal survival and transformation to the secretory decidual phenotype.