AIP Conference Proceedings 2193, 040007 (2019); https://doi.org/10.1063/1.5139369 2193, 040007
© 2019 Author(s).
High progesterone levels on the day of hCG administration in controlled ovarian hyperstimulation procedures reduce the
expression of progesterone receptors in the endometrium Macaca nemestrina
Cite as: AIP Conference Proceedings 2193, 040007 (2019); https://doi.org/10.1063/1.5139369 Published Online: 10 December 2019
Nurhuda, Rosalina Thuffi, Dwi Ari Pudjianto, et al.
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High Progesterone Levels on the Day of hCG Administration in Controlled Ovarian Hyperstimulation Procedures Reduce
the Expression of Progesterone Receptors in the Endometrium Macaca nemestrina
Nurhuda
1,a), Rosalina Thuffi
4,b), Dwi Ari Pudjianto
1, R.Muharam
2, Ninik Mudjihartini
3, Andhea Debby Pradhita
4, Kusmardi Kusmardi
51Departement Biology Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430
2Department Obstetrics and Gynecology, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430
3Departement Biochemistry Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430
4Biomedical science, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430
5Departement Pathology Anatomy, Faculty of Medicine, Universitas Indonesia, Jl Salemba Raya No 6, Senen, Central Jakarta 10430
Corresponding author: a)[email protected]
Abstract. Progesterone (P4) increases on the day of hCG administration in Controlled Ovarian Hyperstimulation (COH) procedure has reduced the rate of pregnancy in Assisted Reproductive Technology (ART). The aim of the study was to analyze the effect of the increase in P4 on the day of hCG administration to the expression of progesterone receptor (PR).
Method: 15 samples of Macaca nemestrina endometrial tissue, in the form of stored biological materials, originated from groups of natural cycles and cycles stimulated in the COH procedure. For 16 endometrial tissue samples, PR expression was measured by immunohistochemical (CPI) technique. The level of PR expression was compared between the natural cycle and the stimulated cycle that got P4≥1 ng/mL and P4 <1 ng/mL. Results: PR expression was detected in three endometrial compartments, which were glandular cells, epithelial luminal cells and stroma. Semi-quantitative analysis used the formula H_SCORE = Σpi (i + 1). PR expression in the gland epithelium from natural cycle group and stimulated cycle with P4≥1 ng / mL and P4 <1ng / mL respectively 2.85 vs, 2.82 vs 2.43. The Kruskal Wallis test, PR expression in the glandular epithelium in the three groups was statistically different (P <0.05). PR expression in and between P4 <1 ng / mL with P4≥1 ng / mL in the stimulated cycle, it did not differ statistically (P > 0.05). Then, PR expression between P4 in the natural cycle with P4 <1 ng / mL in the stimulated cycle there was no significantly different (P > 0.05). PR expression in the luminal epithelium and stroma, between natural cycles and stimulated cycles was also not significantly different (P>
0.05). Conclusion, the level of P4 > 1 ng / mL on the day of administration of hCG in the COH procedure decreases PR expression in the endometrial gland.
Keyword: P4 on the day of hCG administration, COH, endometrial receptivity, progesterone receptor.
INTRODUCTION
Controlled ovarian hyperstimulation (COH) is the procedure in assisted reproductive technology (ART) that is needed for supporting follicular development, therefore the woman hopefully could have more than one follicle in one menstruation cycle. This cycle named stimulated cycle. Increased levels of progesterone (P4) at the end of the follicular phase or on the day of hCG administration during the COH procedure occur often, although emphasis has been made through GnRH injection 1. The cause of the increase in P4 levels is due to the production of many follicles, where each granulose follicle cell contributes to the secretion of the P4 hormone. In the case of high P4 levels on the day of hCG administration is an indicator of success of the COH procedure 2. However, many studies had reported that high P4 levels on the day of hCG administration negatively affects endometrial maturation 3-9, therefore that the pregnancy rate falls10-16.
In the stimulated cycle, endometrium exposed to supra-physiological steroid hormones causes asynchronous maturation with stromal morphology 17,18. Previous research also reported that COH caused changes in the expression of several endometrial receptivity (ER) biomarkers in the endometrial gland epithelium17,19-21.
In a natural cycle, endometrial maturation is controlled by steroid hormone responses, especially P4. Adequate P4 hormone response modulates the transformation of endometrial tissue and maintains an environment suitable for implantation. The mechanism action of P4 in the endometrium is mediated by specific intracellular receptors. The interaction of the P4 with its specific receptors activates certain genes, therefore that the endometrium become receptive during the implantation period. The high P4 concentration on the day of hCG administration resulted in an asynchronous embryo with the endometrium, which would interfere with the implantation process 22.
There is little information related to the impact of increased P4 on endometrial receptivity during the COH procedure, but there have been many reports of the effect on the pregnancy rate. The concentration of P4 that more than 1 ng/mL has been reported reduce the rate of pregnancy23-26. In this study, we examined the impact of the high concentration of P4 (P4≥1ng/mL) on the day of hCG administration against the expression of PR. The aim was to evaluate whether the high concentration of P4 (P4≥1ng/mL) on the day of hCG administration had a negative impact on PR.
METHODS Animals
Animals used in this experimental were females Macaca nemestrina at reproductive age, 8-10 years and weigh 5- 8 kg that had history of given birth. The animals were obtained from the Primate Animal Study Center, Bogor Agricultural University, Bogor, Indonesia. The study protocol was approved by the Institutional Animal Care and Use Committee for Primate Animal Studies, Bogor Agricultural Institute.
The animals chosen for use in this study were tattooed with identification numbers in the growing area and placed in individual cages that made of stainless material in the room. All animals were quarantined and adapted to new individual cages for two to three menstrual periods. During this time, animal health was maintained, and any care was given as needed.
Controlled ovarian hyperstimulation procedure (COH)
For the COH procedure, a combination of gonadotrophin was given with the long GnRH protocol using one of the following three regimens: 1. Recombinant follicle stimulating hormone (rFSH) Gonal F; Merck KGaA, Darmstadt, Germany), 2. GnRH agonist (suprefact), Sanofi S.A., Paris, France). 3. hCG (Pregnyl; Merck KGaA). The GnRH agonist administered at a dose of 160 μg began in the luteal phase in the middle of the previous menstrual cycle and continues until the day before ovulation (about 14 days). After getting the hormone estradiol < 70 pg / mL on the second day of menstruation, administration was combined with rFSH at doses of 30, 50 and 70 IU for the three treatment groups. Recombinant FSH was injected on the second day after menstruation at doses according to the treatment group for 10 days, until the estradiol secretion peaked. Furthermore, hCG was given at a dose of 10,000 IU, equivalent to 3,200 IU. The luteal phase was determined by measuring serial progesterone levels that begin on the postovulatory day.
Endometrial collection
The samples in this experimental were stored endometrial tissue from the Macaca nemestrina. The uterus of each animal was collected 9-10 days after the peak of estradiol secretion in the normal menstrual cycle group and stimulated groups. Before surgery, each animal was anesthetized with ketamine at a dose of 0.1 mL/kg body weight. In necropsy, the uterus was rinsed with phosphate buffer, and part of the tissue is incubated in 10% formalin solution and then implanted in paraffin.
Hormones assay
Serum P4 on the day of hCG administration was determined by a competitive chemiluminiscent immunoassay (Immulite, DPC, Los Angeles, CA, USA). The sensitivity of this method is 0.2 ng / mL and the interassay variation coefficient is 6.7%. The blood was left frozen, and the serum was separated and stored at 20 ° C until tested.
Hematoxylin Eosin staining
The hematoxylin-eosin staining procedure started from the deparaffinized part using xylol and then re-hydrates using alcohol and was washed in running water. Preparates were put into hematoxylin (Meyer solution), rinsed with running water and dipped into saturated lithium carbonate, after that rinsed with running water for 5 minutes.
Immunohistochemistry for progesterone receptors.
Endometrial tissue in paraffin blocks were cut thin, measuring 0.4-0.5 um, then affixed to the glass object that had been coated with APEX. The paraffinization process was carried out in xylol solution and continued with dehydration in an alcoholic solution, then washed in a 0.05 M PBS solution pH 7.2. Next, incubated for 10-15 minutes in H2O2
solution and washed with water. The slides were entered in the retrieval buffer solution and heated for 30 minutes in Retrieval Generation One (GR1) for 30 minutes. After cooling, the slides washed with running water and added progesterone anti-receptor monoclonal antibody (clone Sp2, Biogear) dilution of 200 x and incubated 60 minutes of room temperature. Then the slide washed with buffer in solution, added polymer HRP and incubated for 25 minutes at room temperature. The slides washed again in PBS solution. Then, one drop of dye added on the slide and leave it for 5 minutes then washed again with running water. The slides then incubated for 1 minute in HE coloring and washed again with running water. Then, the slide closed with mounting media and observed under a light microscope.
Ca mamae tissue used as positive control and negative control, which is an endometrial tissue that is not reacted with the marker being examined. Slides were seen under light with 1000 x magnification and randomly selected five areas and photographed using a camera. Calculation of the number of cells and the intensity of the brown color that appeared was calculated in five areas using the J Image Analyzer programme.
The measurement was done semi-quantitatively using the formula H_SCORE = = Σpi (i + 1), Pi is the percentage of colored cells (value 0-100%) i is the color intensity. Strong intensity was given a positive score of +3, moderate intensity, score + 2, weak intensity rated +1 and 0 no cells were colored. The value for each preparation was the average number of positive coloring values and color intensity.
Statistical analysis
Concentration of P4 was found not in normal distribution. Therefore, data were analyzed by non-parametric analysis by Kruskal-Wallis using SPSS 23.0. P-value < 0,005 was considered significant.
RESULTS
Concentration of P4 within group
Concentration of P4 from 15 samples were analyzed by compared them within dose groups and the control group.
Figure 1 shows that samples stimulated by 30IU rFSH, 50IU rFSH, and 70IU rFSH had no significant different of P4 concentration (p value > 0,005).
FIGURE 1. Concentration of P4 in natural cycle as control group and in stimulated cycle. Concentration of P4 within gorup found no significant different with p-value 0,146. It was analyzed by Kruskal-Wallis test.
The location of PR expression in Macaca nemestrina endometrium
The expression of PR was examined by immunohistochemical on Macaca nemestrina endometrial tissue mid luteal phase. Positive values measures were based on the brown color that seen in the nucleus. Five-teen samples examined were tested positive with different levels of expression. The expressions were detected in nuclei of the glandular epithelium, luminal epithelium and endometrial stromal cells (Figure 2).
FIGURE 2. Immunohistochemistry of PR expression in Macaca nemestrina endometrial compartment. (a) Natural cycle, (b) stimulated cycle with P4 <1 ng/mL, (c) stimulated cycle with P4≥1 ng/mL, (d) positive control, (e) negative control, (f) staining
HE for endometrial tissue. Original magnification of (a) to (e) was 40x, and (f) was 10x.
(d) (f)
(b) (c) (a)
Kelenjar Stroma
Epitel luminal
(e)
H-score of PR expression in endometrial Macaca nemestrina
In this study, we compared the value of H score expression in the glandular epithelium, luminal epithelium and endometrial stroma between natural cycles and stimulated cycles. The result of P4 concentration within group in figure 1 found no significant different. Therefore stimulated cycles than did not divided by rFSH group, but by concentration of P4, P4<1 ng/mL and P4≥1 ng/mL.
Figure 3 shows that in glandular epithelial cells, expression of PR was significant different within concentration P4<1ng/mL, P4≥1ng/m, and the control group (p value < 0,05). PR expression in the luminal compartment and stroma did not show a significant difference within the control group and the stimulated cycle that divided into P4<1 ng/mL and P4≥1 ng/mL (P> 0,05).
FIGURE 3. The average of Hscore PR expression. (a) Hscore of PR expression in glandular, (b) Hscore of PR in luminal, and (c) Hscore of PR in stroma. Significant different of PR expression only found in glandular within group of P4<1ng/mL, P4≥1ng/mL, and control group (p-value 0,015). Both in luminal and stroma, PR expression did not significant different with p-
value 0,113 in luminal and 0,958 in stroma.
DISCUSSION
Early studies have explained that the increase in P4 levels measured on the day of hCG administration during the COH procedure had a negative impact on endometrial reception, which will reduce the rate of pregnancy. A study shows that 84% of non-pregnant patients occur with P4 1.3 ng/mL and only 5% of them were pregnant [27]. However, there is no agreement on the ideal of P4 concentration as an indicator for optimal endometrial development leading to the receptive stage. The results of the previous studied found that P4>1ng/mL on the day of hCG administration could reduce the rate of pregnancy [10-17]. This fact certainly indicates that there might be an endometrial receptive disorder due to an increase in P4 concentration on the day of hCG administration.
* (a) (b)
(c)
This study focused on the level of PR expression in Macaca nemestrina endometrium as an indicator of its progress towards receptive stages. The use of female primate animals as a model for research on assisted reproductive technology in humans has been widely carried out. This is based on the similarity of reproductive physiology with human chemistry such as neuroendocrine control, ovarian cyclic function, reproductive tract, placental formation during pregnancy, and reproductive aging from puberty to menopause [28-30].
An increase in P4 levels on the day of hCG administration or premature luteinization (PL) often occurs in the COH procedure. In this experiment, 36% samples were obtained P4 levels more than 1 ng/mL (data not shown) after administration of recombinant FSH (Gonal-F) doses of 30 IU, 50 IU and 70 IU at the COH protocol. From our result, this increase in P4 levels was not caused by high-dose rFSH. It was suspected that this case arises from an increase in the sensitivity of LH receptors from granulosa cells to FSH and ovarian responses [31]. Some research showed that number of antral follicles of less than 6, characterized by poor response and the number of antral follicles 12 was a normal response and the number of antral follicles of more than 13 was a high response [32]. The mechanism of adverse effects of increased P4 was the abnormally accelerated maturation of the endometrium which leads to impaired endometrial reception.
We examined the expression of PR in 15 Macaca nemestrina endometrial samples to evaluate the adverse effects of the increased P4. In immunohistochemical, PR expression was detected in the nuclei of glandular epithelial cells, luminal epithelium and stroma. The expression pattern in the three endometrial compartments was seen strong in the nucleus of the luminal glandular and epithelial cells and was weak in stromal cells. This finding had similarity to previous studies, where expression of PR was detected in the nuclei of luminal and epithelial glandular epithelial cells and stroma [33-34]. Based on this result, it was believed that P4 was a very important regulator for the development of endometrial reception through its activity with the PR in these three compartments and its failure may be related to infertility problems in women [34-36].
Significant reduction of PR expression found in the nucleus cell of glandular epithelial within group (P4≥1 ng/mL;
P4<1 ng/mL; and natural cycle) (p<0.05). This result was related with the theory that proliferation and differentiation of glandular epithelial as the surface of endometrium correlated with success implantation procedure. Progesterone
≥1 ng/mL after COH disturbed the PR expression by made it lower than the normal level suspected as a result of internalization. The expression of PR in the luminal epithelium and stroma also decreased, but was not statistically significant. There seems to be a different in response in to P4 on the day of hCG administration in each endomentrial compartment [17,37,38].
There were morphologically and histologically differences in high P4 levels compared to normal P4 levels [39].
The endometrium that undergoes an advanced maturation process more than 3 days during the COH procedure, there was no pregnancy. The accelerated maturation of the endometrium causes the window of implantation to open in a short period and had interfered with the implantation process and pregnancy [40,41]. The effects of high P4 on hCG administration days on endometrial development had also been investigated. Labarta et al., 2011 found 140 peri- implantation endometrial genes significantly dysregulated with high P4 (P4> 1.5 ng/ml). Van Vaerenbergh et al., 2011 found a significant change in global gene profile in the endometrium of IVF patients with P4 levels> 1.5 ng/mL on the day of hCG administration [42,43].
Many studies have reported that the use of a high P4 threshold between 0.5 - 2 ng/mL has been shown to reduce pregnancy rates, but the mechanism cannot be explained yet [12,14,42]. According to the data we obtained, the PR expression of P4<1 ng/mL on day of hCG administration in a stimulated cycle are almost similar to natural cycles.
Therefore, cycle with stimulation still could do the embryo transfer procedure in IVF if the P4 level after COH is less than 1 ng/mL.
The limitation of this study was that the number samples are small, ideally 5 each for repetition, which disrupts statistical power. In addition, the experimental response to recombinant FSH stimulation did not correlate with the magnitude of the rFSH dose given. Although there was a decreased in PR expression in the luminal epithelial region, significant differences of H-score value resulted from immunohistochemistry test were difficult to obtain compared to other techniques. The strength of our research was that the uterine organ was taken at the same periode of time so that it could be compared between natural cycles and stimulated cycles.
In conclusion, the P4 level ≥1 ng/mL on the day of hCG administration in the COH procedure decreases PR expression in the glandular epithelial compartment and the middle of the secretion phase. Therefore, the use of frozen embryos to be transferred in the next menstrual cycle without COH is an option to get higher success rate.
ACKNOWLEDGMENTS
We would like to thank for Director of Research and Public Services Universitas Indonesia for PITTA research program 2018.
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