Concentrations of endometrial protein PP 14 and CA-125 in uterine flushings performed in natural and stimulated cycles

Ernest Hung Yu Ng1,4, Susan Margaret Laird2, Tin Chiu Li3, William Shu Biu Yeung1 and Pak Chung Ho1

1 Department of Obstetrics and Gynaecology, The University of Hong Kong, Hong Kong Special Administrative Region, People’s Republic of China, 2 Division of Biomedical Sciences, Sheffield Hallam University, City Campus, Sheffield and 3 Centre for Reproductive Medicine and Fertility, The Jessop Wing, Sheffield Teaching Hospitals NHS Trust, Sheffield, UK

4 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, The University of Hong Kong, 6/F, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong. e-mail: nghye{at}hkucc.hku.hk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Impaired implantation in assisted reproduction cycles with high serum estradiol (E2) concentrations may be attributed to abnormal endometrial development. This study compared concentrations of endometrial proteins in uterine flushings of infertile patients between natural and stimulated cycles. METHODS: Patients received a standard regimen of ovarian stimulation. Seven days after the LH surge in natural cycles or the hCG injection in stimulated cycles, uterine flushings were performed by slowly injecting and aspirating normal saline through a paediatric Foley catheter. Natural cycles were considered as group A whereas stimulated cycles with serum E2 <20 000 pmol/l and serum E2 >20 000 pmol/l were classified as groups B and C respectively. PP 14 and CA-125 in uterine flushings were measured and expressed per total protein content. RESULTS: Concentrations of the total protein, PP 14 and CA-125 in the uterine flushings were similar among the three groups. PP 14 per total protein in the uterine flushings was significantly correlated with serum E2 on the day of hCG (r = 0.459; P = 0.009) in natural cycles only but not in stimulated cycles. CONCLUSION: There was no significant difference between natural and stimulated cycles in concentrations of PP 14 and CA-125 in uterine flushings performed in the mid-luteal phase.

Key words: CA-125/excessive responders/PP 14/uterine flushing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
High serum oestradiol (E2) concentrations resulting from excessive ovarian responses adversely affect the outcomes of assisted reproduction cycles (Forman et al., 1988Go; Simón et al., 1995Go, 1998; Check et al., 2000Go; Ng et al., 2000aGo), although such adverse effects were not observed in some studies (Chenette et al., 1990Go; Gelety and Buyalos, 1995Go; Sharara and McClarmrock, 1999Go; Papageorgiou et al., 2002Go). We have demonstrated significant impairment in pregnancy rates during stimulated IVF cycles when the serum E2 concentra tion on the day of hCG was >20 000 pmol/l (Ng et al., 2000aGo). Oocyte quality was not affected by the extremely high E2 concentrations as surplus embryos from cycles with different E2 concentrations had comparable implantation and pregnancy rates in frozen–thawed transfer cycles. A further study (Ng et al., 2003Go) of only ICSI cycles for severe male factors confirmed that both oocyte and embryo quality were not compromised by the excessively high E2 concentrations.

Based on these results, we postulate that reduced implantation in cycles with high serum E2 concentrations is most likely related to an adverse environment in the endometrium. The impairment in uterine receptivity of the excessive responders is not explained by a rapid decline in serum E2 around the peri-implantation period (Ng et al., 2000bGo) but may be attributed to abnormal endometrial development in the form of delayed glandular maturation and advanced stromal morphology (Basir et al., 2001aGo). Colour Doppler ultrasound examination also revealed that the number of women showing endometrial colour signals was significantly lower in excessive responders despite much lower flow indices of uterine vessels, when compared with moderate responders, i.e. serum E2 <20 000 pmol/l (Basir et al., 2001bGo). The above findings in the peri-implantation endometrium of the excessive responders prompted us to investigate any change in endometrial function following excessive ovarian stimulation.

The aims of this study were to measure and compare endometrial placental protein 14 (PP 14, also known as glycodelin-A) and CA-125 concentrations in uterine flushings of IVF patients with natural cycles and those with stimulated cycles with serum E2 concentration on the day of hCG <20 000 and >20 000 pmol/l. Uterine flushing measurements of these proteins have been used to investigate endometrial function in a number of previous studies (Dalton et al., 1995Go; Li et al., 1998Go; Okon et al., 1998Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infertile patients attending the Assisted Reproduction Unit at Department of Obstetrics and Gynaecology, University of Hong Kong, for IVF treatment were recruited when embryo transfer was not performed as a result of no sperm retrieved in testicular sperm extraction, absence of fertilization due to male factors or serum E2 levels on the day of hCG >20 000 pmol/l. All fresh embryos were cryopreserved in patients with serum E2 levels on the day of hCG >20 000 pmol/l because of associated risks of ovarian hyperstimulation syndrome and reduced pregnancy rate in these stimulated cycle. Patients on the waiting list for IVF treatment who had regular ovulatory cycles were also recruited in natural cycles for comparison. Every patient gave a written informed consent prior to participating in the study, which was approved by the Ethics Committee, Faculty of Medicine, University of Hong Kong. Each patient was evaluated only once during the study period and did not receive any monetary compensation for study participation.

Stimulated cycles
The details of the long protocol of ovarian stimulation regimen at our centre have been previously published (Ng et al., 2000aGo). In short, all women were pre-treated with buserelin (Suprecur; Hoechst, Germany) nasal spray 150 µg four times a day from the mid-luteal phase of the cycle preceding the treatment cycle and received hMG (Pergonal; Serono, Switzerland) for ovarian stimulation. hCG (Profasi; Serono) 10 000 IU was given i.m. when the leading follicle reached 18 mm in diameter and there were at least three follicles >15 mm in diameter. Blood was taken on the day of hCG for serum E2 concentrations.

Natural cycles
These patients had not received any steroidal hormones for ≥2 months prior to the study cycle and used condoms for contraception during the study cycle. They were asked to attend the clinic daily from 18 days before the next expected period for serum E2 and LH concentrations until the LH surge, which was defined when the serum LH level was more than double the mean of the preceding values.

Uterine flushing
All uterine flushings were performed 7 days after the hCG injection in stimulated cycles or the LH surge in natural cycles as an outpatient procedure as previously described (Li et al., 1993aGo). A bivalve speculum was inserted into the vagina and the cervix was cleaned with a piece of gauze soaked with normal saline. Then a size 8 Foley catheter was introduced into the uterine cavity and the balloon of the catheter was inflated with 1 ml of normal saline. Following this, 1 ml of normal saline solution was gradually flushed into the uterine cavity via the opening connected to the inner lumen; afterwards gentle suction via the opening was applied to recover the fluid. The flushing was carried out slowly over a period of 15 s. The procedure was repeated five times, each time using a fresh sample of 1 ml of normal saline solution. A total of 5 ml of normal saline was used to carry out the flushing. The samples from a patient were pooled, centrifuged and stored at –20°C for PP 14 and CA-125 assay. Blood was also taken for E2 and progesterone concentration measurements on the same day of uterine flushing.

PP 14, CA-125, total protein in uterine flushings and serum hormonal assays
PP 14 was measured by radioimmunoassay using the method previously described (Bolton et al., 1983Go). In brief, PP 14 was iodinated by the chloramine-T method and the resulting tracer was purified using a column of Con-A Sepharose. For this assay, 100 µl of standards prepared from purified PP 14 were incubated with 100 µl of antisesrum, at a dilution to bind 45% of the added tracer. The antibody-bound tracer was separated from the unbound tracer using Amerlex-M magnetic separating reagent (Amershan International, UK). The sensitivity of the assay was 3 ng/ml, and the intra- and inter-assay coefficients of variation (CV) were <10%.

Serum CA-125 was measured using a commercially available kit (Automated Chemiluminescence System; Bay Corporation, USA). The sensitivity of this assay was 1.7 IU/ml and samples with CA-125 levels >1000 IU/ml were automatically diluted by the system. The intra- and inter-assay coefficients of variation were 4.7 and 5.5% respectively at high CA125 level (mean: 875 IU/ml).

Concentrations of PP14 and CA125 were also expressed in relation to the total protein content in the uterine flushing, which was measured by a colorimetric method (Coomassie® Plus-200 Protein Assay; Pierce Biotechnology Inc., USA).

Serum E2, LH and progesterone concentrations were measured using commercially available kits (Automated Chemiluminescence System). The intra- and inter-assay CV were <10% for these hormonal assays.

Statistical analysis
Natural cycles were considered as group A whereas stimulated cycles with serum E2 <20 000 pmol/l and serum E2 >20 000 pmol/l were classified as groups B and C respectively. The primary outcome measures were PP 14 and CA-125 concentrations in the uterine flushings. Continuous variables were not normally distributed and were given as median (range), unless otherwise indicated. Statistical comparison was carried out by Kruskal–Wallis, {chi}2 and Mann–Whitney tests, where appropriate. Correlation was assessed by the Spearman rank method. Two-tailed P < 0.05 was taken as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 107 consecutive patients was included: 36 patients in group A, 35 patients in group B and 36 patients in group C. Table I summarizes the demographic data, ovarian responses and endometrial thickness of the three groups. There were no differences in age of women, primary/secondary infertility, causes of infertility and duration of infertility between the three groups. Significantly more oocytes were obtained in group C than in group B, despite similar dosage and duration of hMG used in both groups. Endometrial thickness in group C was significantly higher than that in group A, but such differences were not observed between groups A and B or between groups B and C.


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Table I. Comparison of demographic data, ovarian responses and endometrial thickness
 
Although the median PP 14 concentration in flushings from patients in group B was considerably lower than that seen for patients in groups A and C, the range in values was very similar and therefore there was no significant difference in values (Table II). Concentrations of the total protein and CA-125 in the uterine flushings were similar between the three groups, while serum E2 and progesterone concentrations on the day of uterine flushing differed significantly between the three groups.


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Table II. Comparison of PP 14 and CA-125 concentration in uterine flushings and serum hormonal levels on the day of uterine flushing
 
The total protein content in the uterine flushings was significantly correlated with PP 14 (r = 0.475; P < 0.001) and CA-125 concentrations (r = 0.509; P < 0.001) when all cycles were taken together. Significant correlations were also seen in natural cycles and in stimulated cycles. PP 14 per unit protein in the uterine flushings was significantly correlated with serum E2 on the day of the LH surge (r = 0.459; P = 0.009) in natural cycles only. On the other hand, serum E2 on the day of hCG and serum E2 and progesterone on the day of uterine flushings were not correlated with concentrations of total protein, PP 14 and CA-125, nor with PP 14 and CA-125 per unit protein in stimulated cycles and in all cycles.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The success of implantation depends upon a perfect dialogue between good quality embryos and a receptive endometrium. The endometrium in the follicular phase undergoes a progressive increase in thickness due to hyperplasia of both glandular and stromal elements under the stimulation of rising serum E2 concentration as a result of follicular development. Under the influence of progesterone, the endometrium undergoes sequential events including maturation, secretory transformation of the glandular elements, edema formation, vascular proliferation and decidualization of the stroma in the luteal phase. Therefore, endometrial receptivity can be evaluated by ultrasound examination of the endometrium, histological evaluation of an endometrial biopsy and endometrial proteins in uterine flushing.

Uterine flushing provides a reliable and non-invasive tool to evaluate endometrial function by measuring endometrial proteins, which may be important for the process of implantation. Pellicer et al. (1989Go) first used a paediatric Foley catheter and 10 ml of normal saline solution to flush out the contents of the uterine cavity for the total protein concentration in eight women with premature ovarian failure receiving HRT. We have used extensively this method of uterine flushing to study the endometrial function and the amount of discomfort was significantly less than that of endometrial biopsy (Li et al., 1993aGo). Moreover, the complications including vaginal bleeding, pelvic infection and delayed abdominal pain were not encountered. Other techniques of obtaining uterine fluid include direct aspiration through a paediatric feeding tube (Abaé et al., 1992Go) and an intrauterine microdialysis device consisting of microdialysis tubing glued into a balloon catheter on one side and into a polypropylene tube on the other (Licht et al., 1998Go).

In previous studies, the loss of flushed normal saline through the Fallopian tubes was 1–2 ml in six (6.7%) out of 90 consecutive cases and 3–5 ml in another three (3.3%) (Li et al., 1993aGo) when the flushings were performed during the luteal phase using a Foley catheter. On the other hand, Hamilton et al. (1998Go) showed that the recovered volume of the flushings done in the proliferative phase depended on the patency of the Fallopian tubes and varied significantly between serial samples obtained from the same individual and between different cycle days. They proposed to use concentration of endometrial proteins after correction for total protein content because the concentrations of the endometrial protein (PP 14) were better correlated with the total protein than with the recovered volume. In this study, all flushings were performed in the mid-luteal phase but the recovered volume of normal saline for each patient was not recorded. In order to account for possible variations in the recovery of the flushed fluid, concentrations of PP 14 and CA-125 corrected for total protein content were also given.

Two endometrial proteins, i.e. PP 14 and CA-125, have been extensively evaluated in the literature. PP 14 has been renamed glycodelin-A after the discovery of a glycoprotein with a well-defined amino acid sequence and unique oligosaccharide structure, which reacted with antibodies against human PP 14 (Dell et al., 1995Go). We continued to use the term ‘PP 14’ in this study as our PP 14 assay has not been validated for the detection of the specially sequenced glycodelin-A. PP 14 possesses immunosuppressive activity (Bolton et al., 1987Go; Pockley et al., 1988Go; Okamoto et al., 1991Go) which may facilitate successful implantation and maintenance of pregnancy by preventing the rejection of the foreign fetal allograft. PP 14 levels in uterine flushings were correlated with endometrial dating and volume fraction measurement of the glands during the natural cycles (Li et al., 1993bGo). Moreover, the presence of detectable concentrations of PP 14 in uterine flushings was significantly associated with normal histological dating.

CA-125 is a glycoprotein identified by monoclonal antibody OC-125 and has been demonstrated in the epithelium of endometrium and Fallopian tubes and in the mesothelial cells of the peritoneum. Serum CA-125 has been extensively studied in the diagnosis and monitoring of epithelial ovarian carcinoma. CA-125 is an exocrine product of endometrial glands, exclusively from epithelial cells (Weintraub et al., 1990Go). Uterine fluid contains a high concentration of CA-125 but its roles in implantation remain to be elucidated. PP 14 in the uterine flushings is better than CA-125 as a marker of endometrial morphology in women with recurrent miscarriages (Dalton et al., 1995Go) and with peri-/post-menopausal bleeding (Li et al., 1998Go).

To the best of our knowledge, this is the first study to measure PP 14 and CA-125 concentrations in uterine flushings of infertile patients during their stimulated cycles and to compare concentrations of these two endometrial proteins among natural, moderately stimulated and excessively stimulated cycles. We found that concentrations of total protein content, PP 14 and CA-125 in the uterine flushings were not affected by ovarian stimulation, whether the stimulation was moderate or even excessive. This finding is not consistent with our previous observation of abnormal endometrial glandular development in excessive responders (Basir et al., 2001aGo). Using morphometric analysis, we demonstrated a trend of progressive decrease in the size of glandular volume and luminal diameter of peri-implantation endometrium with increasing serum E2 concentrations from natural to excessively stimulated cycles. The excessive responder group had a significantly higher number of sub-nuclear vacuoles but a lower percentage of moderate to abundant filling of endometrial glands when compared with the moderate responder group.

Glycodelin is absent from the endometrium during the peri-ovulatory phase, but is synthesized in this tissue during the peri-implantation phase in response to progesterone and relaxin exposure and is abundant during the last week of the luteal phase (Seppala et al., 2001Go). Brown et al. (2000Go) also found a significantly increased proportion of glycodelin-stained endometrial cells in 15 oocyte donors undergoing ovarian stimulation when compared with 19 natural-cycle control patients. In this study, endometrial biopsies were not available for comparison with the findings in uterine flushings.

The timing of uterine flushings may explain why we cannot find any difference in concentrations of PP 14 and CA-125 in uterine flushings after ovarian stimulation. Concentrations of both PP 14 and CA-125 in uterine flushings have been shown to increase in a similar pattern throughout the luteal phase of a natural cycle, with the most marked increase occurring 6 days after the LH surge (Dalton et al., 1995Go). Similarly, the expression of glycodelin in the endometrial glands was increasing throughout the late luteal phase of natural and stimulated cycles (Brown et al., 2000Go). This study was designed to examine the peri-implantation period and therefore all uterine flushings were performed 7 days after the LH surge or the hCG injection. It is possible that uterine flushings performed later in the cycle (e.g. 10–12 days after the LH surge or the hCG injection) may be better to detect significant differences in concentrations of PP 14 and CA-125 between natural and stimulated cycles. Concentrations of PP 14 and CA-125 in uterine fluid can vary over a large range in individual women (Abaé et al., 1992Go; Dalton et al., 1995Go) and therefore our cross-sectional study may not reveal any difference in the concentrations of endometrial proteins in uterine flushings after ovarian stimulation. A longitudinal study of both natural and stimulated cycles from the same patient may be a more appropriate study design.

In uterine flushings, PP 14 concentrations are reduced in patients with unexplained infertility (Mackenna et al., 1993Go) while CA-125 concentrations are independent of the cause of infertility (Hamilton et al., 2002Go). We have very few cases of unexplained infertility in this study and each group had similar causes of infertility. Therefore, it is very unlikely that the different causes of infertility in each group can mask the difference in PP 14 and CA-125 concentrations in uterine flushings after ovarian stimulation. The endometrial lining of the excessively stimulated group was thicker than that of the natural cycle group. This supports our previous observation of increased endometrial thickness in stimulated cycles when compared with natural cycles (Basir et al., 2002Go).

When only natural cycles were considered, PP 14 per unit protein was significantly correlated with serum E2 level on the day of hCG. However, such correlation was not found in stimulated cycles, whether moderate and excessive responders were separated or not. This suggested that the secretion of PP 14 by endometrial glands might reach its maximum during the natural cycles and ovarian stimulation may not further increase its concentration in the uterine flushing. Similarly, Brown et al. (2000Go) showed a significant positive correlation between endometrial glycodelin expression and serum E2 levels in natural cycles only. There was again no significant correlation between serum E2 or progesterone levels and endometrial glycodelin expression in stimulated cycles.

Serum concentrations of PP 14 and CA-125 were not measured in this study because serum PP 14 concentrations were not predictive of histological dating or morphometric analysis (Li et al., 1993bGo) and serum CA-125 in women undergoing ovulation induction were mainly derived from the ovaries (Gurgan et al., 1993Go). However, the serum measuremements may have been useful since serum PP 14 concentrations during the natural cycle were shown to reflect endometrial receptivity to implantation and pregnancy during successive assisted reproduction cycles (Westergaard et al., 1998Go, 2003). Locally secreted cytokines from the endometrium are important in controlling the implantation process. Leukaemia inhibitory factor and tumour necrosis factor present in the uterine flushings during the mid-luteal phase were predictive of implantation in the subsequent IVF cycles (Lédéé-Bataille et al., 2002Go). In the recurrent implantation failure group, the matrix metalloproteinase score and interleukin (IL)-1{beta} concentrations of uterine fluid were significantly higher than those in the control fertile group, whereas concentrations of interferon-{gamma} and IL-10 were significantly lower (Ingaki et al., 2003Go). However, these locally secreted cytokines may not be detectable in uterine flushings because of the small volume of fluid recovered and the very minute amounts present in the flushings (Lédéé-Bataille et al., 2002Go; Olivennes et al., 2003Go).

In conclusion, no significant difference was found between natural and stimulated cycles in concentrations of PP 14 and CA-125 in uterine flushings performed in the mid-luteal phase. It would appear that the adverse impact of high serum estradiol concentration on endometrial function in the peri-implantation period is not associated with alterations in the PP 14 and CA-125 concentrations in the uterine flushings.


    Acknowledgements
 
This study was funded by the Hong Kong Research Grant Council (HKU 7341/00M).


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 12, 2003; accepted on January 8, 2004.





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