Rapid screening of infertility-associated gynecological conditions via ambient glow discharge mass spectrometry utilizing urine metabolic fingerprints.

Infertility presents a widespread challenge for many families worldwide, often arising from various gynecological diseases (GDs) that hinder successful pregnancies. Current diagnostic methods for GDs have disadvantages such as low efficiency, high cost, misdiagnose, invasive injury and etc. This paper introduces a rapid, non-invasive, efficient, and straightforward analytical method that utilizes desorption, separation, and ionization mass spectrometry (DSI-MS) platform in conjunction with machine learning (ML) to detect urine metabolite fingerprints in patients with different GDs. We analyzed 257 samples from patients diagnosed with polycystic ovary syndrome (PCOS), premature ovarian insufficiency (POI), diminished ovarian reserve (DOR), endometriosis (EMS), recurrent pregnancy loss (RPL), recurrent implantation failure (RIF), and 87 samples from healthy control (HC) individuals. We identified metabolite differences and dysregulated pathways through dimensionality reduction methods, with the result of the discovery of 7 potential biomarkers for GDs diagnosis. The ML method effectively distinguished subtle differences in urine metabolite fingerprints. We anticipate that this innovative approach will offer a patient-friendly, rapid screening, and differentiation method for infertility-related GDs patients.

Associations of exposure to phthalates and environmental phenols with gynecological disorders.

Phthalates and environmental phenols might be associated with some benign diseases that have been found to be hormone-sensitive. Current knowledge on adverse effects of these chemicals among reproductive women is limited and often controversial. Therefore, the purpose of this study was to investigate the association between the urinary concentration of phthalates and environmental phenols and gynecological disorders from 512 women of reproductive age. The association between chemical concentration and disease in the control and case groups was statistically determined with the questionnaire survey data and measurements using the LC-MS/MS. The results have shown that DEHP metabolites, ethyl paraben and 3,4-DHB showed significant direct associations with leiomyoma and benign ovarian tumors (p < 0.05). We found statistically significant positive relationships between exposure to chemicals (some DEHP metabolites, DHB) and prevalence of gynecologic disorders (p < 0.05). Furthermore, the ORs for leiomyoma associated with these compounds in always user for personal care products (PCPs) was higher than those of sometimes user. High levels of urinary concentrations of these compounds such as DEHP metabolites and parabens and their metabolites showed significant associations with leiomyoma and benign ovarian tumors.

A simplified method to recover urinary vesicles for clinical applications, and sample banking.

Urinary extracellular vesicles provide a novel source for valuable biomarkers for kidney and urogenital diseases: Current isolation protocols include laborious, sequential centrifugation steps which hampers their widespread research and clinical use. Furthermore, large individual urine sample volumes or sizable target cohorts are to be processed (e.g. for biobanking), the storage capacity is an additional problem. Thus, alternative methods are necessary to overcome such limitations. We have developed a practical vesicle isolation technique to yield easily manageable sample volumes in an exceptionally cost efficient way to facilitate their full utilization in less privileged environments and maximize the benefit of biobanking. Urinary vesicles were isolated by hydrostatic dialysis with minimal interference of soluble proteins or vesicle loss. Large volumes of urine were concentrated up to 1/100 of original volume and the dialysis step allowed equalization of urine physico-chemical characteristics. Vesicle fractions were found suitable to any applications, including RNA analysis. In the yield, our hydrostatic filtration dialysis system outperforms the conventional ultracentrifugation-based methods and the labour intensive and potentially hazardous step of ultracentrifugations are eliminated. Likewise, the need for trained laboratory personnel and heavy initial investment is avoided. Thus, our method qualifies as a method for laboratories working with urinary vesicles and biobanking.

Population based study of genital Chlamydia trachomatis prevalence and associated factors in Norway: a cross sectional study.

BACKGROUND: The number of diagnosed cases of Chlamydia trachomatis infection has been increasing in the past years in Norway although the testing rate has been relatively stable. The aim of this study was to measure the prevalence of genital Chlamydia trachomatis in young men and women in one county in Norway and determine associated factors in order to better target preventive measures. METHODS: We mailed to a random sample of 10,000 persons aged 18-25 in Rogaland county a mail-back urine sample kit and a self-administered questionnaire with questions on socio-demographic details, health seeking behaviour and symptoms of and history of sexually transmitted diseases. Associations between current Clamydia trachomatis infection and the above mentioned factors were studied by multiple logistic regression. RESULTS: The response rate among women was 18.9% (930/4923) and 11.9% (605/5077) among men. The prevalence of Chlamydia trachomatis infection was 5.8% (95% CI 4.5-6.8) among women and 5.1% (95% CI 3.8-6.8) among men. For men a greater number of partners during the last year (p for trend < 0.001), and living in a municipality without a local youth clinic increased the odds of infection (OR 8.6, 95% CI 2.2-33.9). For women a greater number of partners during the last year (p < 0.001) and not having consulted a family doctor for STIs (OR 2.1 95% CI 1.1-4.2) were positively associated with infection while not having a previous Chlamydia trachomatis diagnosis decreased the odds of having this infection (OR 0.3, 95% CI 0.2-0.7). CONCLUSION: Our results indicate the importance of having a visible youth clinic in each municipality. It also suggests targeting women who have had a previous Chlamydia trachomatis infection diagnosed before.

Detection of the HE4 protein in urine as a biomarker for ovarian neoplasms.

The HE4 protein is overexpressed in ovarian carcinomas and can be detected in serum by an ELISA with sensitivity similar to CA125 and higher specificity for malignant disease. We now demonstrate that HE4 can also be detected in the urine at a specificity level of 94.4%, including 13/15 (86.6%) with stage I/II and 57/64 (89.0%) with stage III/IV disease and including 90.5% of patients with serous ovarian carcinoma. Assaying serum and urine from the same patients showed similar sensitivity. Our data indicate that measuring HE4 in urine may aid diagnosis and the monitoring of response to therapy.

Predicting bacteriuria in urogynecology patients.

OBJECTIVE: This study was undertaken to determine whether reagent strip testing can predict bacteriuria in urogynecology patients. STUDY DESIGN: All women undergoing urodynamic evaluations from June 1997 to October 2001 were identified by using a computerized database. Urine culture results were compared with reagent strip testing. Significant bacteriuria was defined as greater than 10(5) colony-forming units per milliliter. RESULTS: Bacteriuria prevalence was 8.6% (n = 51). Sensitivity and specificity of nitrites were 0.51, (95% CI, 0.31-0.66) and 0.991, (95% CI, 0.974-0.998), respectively. Blood had a lower sensitivity (0.35, 95% CI, 0.20-0.54) and specificity (0.80, 95% CI, 0.75-0.84). Leukocyte esterase was similar to blood with a sensitivity of 0.28 (95% CI, 0.14-0.45) and specificity of 0.83 (95% CI, 0.78-0.87). No combination of tests offered improved sensitivity or specificity over nitrites alone. CONCLUSION: Nitrite dipstick testing has excellent specificity for bacteriuria in urogynecologic patients. These results support the treatment of women with positive nitrites who are preparing to undergo urodynamics without obtaining culture.

Opportunistic screening for genital chlamydial infection. I: acceptability of urine testing in primary and secondary healthcare settings.

OBJECTIVES: To determine the acceptability of opportunistic screening for Chlamydia trachomatis in young people in a range of healthcare settings. DESIGN: An opportunistic screening programme (1 September 1999 to 31 August 2000) using urine samples tested by ligase chain reaction (LCR). Data on uptake and testing were collected and in-depth interviews were used for programme evaluation. SETTING: General practice, family planning, genitourinary medicine clinics, adolescent sexual health clinics, termination of pregnancy clinics, and women's services in hospitals (antenatal, colposcopy, gynaecology and infertility clinics) in two health authorities (Wirral and Portsmouth and South East Hampshire). Main participants: Sexually active women aged between 16 and 24 years attending healthcare settings for any reason. MAIN OUTCOME MEASURES: Uptake data: proportion of women accepting a test by area, healthcare setting, and age; overall population coverage achieved in 1 year. Evaluation data: participants' attitudes and views towards opportunistic screening and urine testing. RESULTS: Acceptance of testing by women (16-24 years) was 76% in Portsmouth and 84% in Wirral. Acceptance was lower in younger women (Portsmouth only) and varied by healthcare setting within each site. 50% of the target female population were screened in Portsmouth and 39% in Wirral. Both the opportunistic offer of screening and the method of screening were universally acceptable. Major factors influencing a decision to accept screening were the non-invasive nature of testing and treatment, desire to protect future fertility, and the experimental nature of the screening programme. CONCLUSIONS: An opportunistic model of urine screening for chlamydial infection is a practical, universally acceptable method of screening.

Assessment of Chlamydia trachomatis prevalence by PCR and LCR in women presenting for termination of pregnancy.

OBJECTIVES: To determine the prevalence of Chlamydia trachomatis in a patient population presenting for legal termination of pregnancy by polymerase chain reaction (PCR) and ligase chain reaction (LCR), from first catch urine and self administered tampons, and comparing with the traditionally collected endocervical swab tested by both PCR and culture. METHODS: Consecutive women attending for legal termination of pregnancy were screened for chlamydia by patient collected first catch urine and tampon, and physician collected endocervical swab. RESULTS: Of 1175 patients with complete samples, there were 33 (2.8%) in whom chlamydia was detected by two or more assays from one or more sample site. Chlamydia was detected equally well by both PCR and LCR in first catch urine (p = 0.25), tampon (p = 0.5), and endocervical swab (p = 0.5). However, both PCR and LCR were significantly better than culture of an endocervical swab (p = 0.0005) for detection of C trachomatis. CONCLUSION: The prevalence of chlamydia in patients presenting for termination of pregnancy was 2.8%. A simple efficient way of performing screening for chlamydia for women presenting for termination of pregnancy is by first catch urine or tampon, which can be tested by the highly sensitive amplification assays, PCR or LCR.

Schistosomiasis of the female genital tract: public health aspects.

In this paper Gabriele Poggensee, Hermann Feldmeier and Ingela Krantz discuss the public health relevance of female genital schistosomiasis (FGS). Some of the stated hypotheses are supported only by clinical observations and/or circumstantial evidence as valid epidemiological and immunological data of this disease entity are still very scanty. Morbidity caused by the presence of schistosome eggs in the lower and upper genital tract have been almost completely neglected during the past two decades. This has been acknowledged by the WHO and, in 1997, the Gender Task Force of the WHO's Tropical Disease Research Programme (TDR) included FGS in a list of scientific areas that deserve high research priority.

Presence of urokinase-type plasminogen activator receptor in urine of cancer patients and its possible clinical relevance.

High levels of urokinase-type plasminogen activator receptor (uPAR) are expressed in various types of cancer. Recent studies showed that cancer patients may have increased levels of soluble (s)uPAR in their serum. In the present study, we show that urine samples from healthy volunteers contain measurable amounts of suPAR. suPAR/creatinine levels from healthy controls showed only little variation over the day and were even stable during a month of continued monitoring. Importantly, urinary suPAR/creatinine levels were highly correlated with serum suPAR concentrations. Urinary suPAR levels were elevated in patients with different types of cancer. Interestingly, part of the urinary suPAR seemed to be present in a cleaved form, as has been found in tumor tissue extracts. Together with the recently established, cell migration-promoting effect of certain cleaved fragments of suPAR, the present data suggest that the measurement of urinary suPAR and/or its cleaved forms might have clinical implications.

[Systematic home screening for Chlamydia trachomatis infections of asymptomatic men and women in family practice by means of mail-in urine samples]. / Systematische opsporing van infecties met Chlamydia trachomatis bij mannen en vrouwen zonder klachten in de huisartspraktijk met behulp van per post verstuurde urinemonsters.

OBJECTIVE: To determine the prevalence and determinants of Chlamydia trachomatis (CT) infections among asymptomatic men and women in general practice. To determine participation rates in a systematic screening programme in general practice, using home obtained mailed urine samples. DESIGN: Cross-sectional study. METHODS: In 15 general practices in Amsterdam, the Netherlands, a sample of 11,005 persons (5541 women and 5464 men), aged 15-40 were invited to send in a urine sample and a completed questionnaire by mail. The urine samples were tested using the ligase chain reaction for DNA amplification. Patients diagnosed with CT were treated and partner notification was performed. RESULTS: 33% of invited males (1809/5464) and 50% of females (2751/5541) sent in the study material. Older patients participated more frequently than younger patients. Participation rates among persons with a Dutch background were higher than rates among persons from other ethnic groups. In 42 men and 79 women a CT infection was identified (2.3% and 2.9% respectively). Infections were more prevalent in patients from Surinam and the Dutch Antilles and in the age category 21-25 years. Type of health insurance as a proxy measure of socioeconomic status was not an indicator of infection. CONCLUSION: The participation in this systematic screening using mail-sent urine samples was 33% in men and 50% in women. The CT prevalences among asymptomatic men and women were 2.3% and 2.9% respectively.

Schistosomiasis of the lower reproductive tract without egg excretion in urine.

The individual and public health impact of female genital schistosomiasis (FGS) has been studied and FGS as a risk factor for acquiring human immunodeficiency virus is discussed. In a community-based study in Tanzania, 40% of the women of child-bearing age (n=543) showed excretion of Schistosoma haematobium eggs in the urine (median=2.2 eggs/10 ml of urine) and 32% (n=263) had S. haematobium eggs in their cervical tissue. Urinary and genital schistosomiasis coexisted in 62% of the women, but S. haematobium eggs were found in the cervix without detectable egg excretion in the urine in 23%. Only 43% of the FGS cases had hematuria. Since FGS frequently exists in women with scanty or no egg excretion in the urine and because this disease manifestation is a considerable individual and public health hazard in S. haematobium-endemic areas, mass treatment targeted to women of child-bearing age should be considered.

Female genital schistosomiasis due to Schistosoma haematobium. Clinical and parasitological findings in women in rural Malawi.

A total of 51 women with urinary schistosomiasis haematobium were examined in order to identify diagnostic indicators for female genital schistosomiasis (FGS). Patients were selected at random from the outpatient department of the Mangochi District Hospital, Malawi. The medical histories were recorded according to a pre-designed questionnaire and the women were subjected to a thorough gynaecological examination including colposcopy and photographic documentation of lesions. Microscopy of genital biopsies revealed that 33 of the 51 women had S. haematobium ova in cervix, vagina and/or vulva in addition to the presence of ova in urine. The most sensitive diagnostic procedure was beside microscopic examination of a wet cervix biopsy crushed between two glass slides, which revealed 25 of the 33 genital infections. There was a significant correlation between the size of genital lesions and the number of ova counted per mm2 of crushed tissue. Women with FGS had significantly more tumours in the vulva than women with schistosomiasis limited to the urinary tract. Most of the observed genital pathology could easily be identified by the naked eye, but colposcopic examination yielded valuable additional information like the demonstration of neovascularisation around cervical sandy patches. Few of the symptoms previously regarded as indicators for FGS could be linked to the presence of schistosome ova in genital tissue. Husbands of infertile women with FGS had children with other women significantly more often than husbands of women who only had urinary schistosomiasis. This, together with the finding that the majority of the divorced women had FGS, indicates that the manifestation of this disease may have implications for the marital and sexual life of the affected women.

Urine reagent strips for diagnosis of schistosomiasis haematobium in women of fertile age.

Hematuria, proteinuria and leukocyturia were semiquantitatively assessed by reagent strips in single morning urine of women of fertile age visiting the outpatient department of the Mangochi district hospital, Malawi. This was part of a diagnostic approach to female genital schistosomiasis (FGS). In 51 women ova of Schistosoma haematobium were detected in urine by a filtration technique. In 33 of these women ova were also present in genital tissue as demonstrated by microscopic examination of biopsies. In 209 women no ova were found in the single urine filtered. There were significantly higher scores for hematuria, proteinuria and leukocyturia as well as of the combined reagent strip index (RSI) in egg-excreting than in egg-negative women. The sensitivity of a single hematuria, proteinuria and leukocyturia reading was 98, 84 and 73%, respectively. However, the respective specificity was only 24, 22 and 23%. The best prediction of urinary schistosomiasis was achieved by a +2 score for hematuria, of which the sensitivity was 94% and the specificity was 61%. The high false-positive rates can probably be explained by contamination of urine by vaginal secretion. Moreover, cases of schistosomiasis have probably been overlooked because only a single morning urine sample was examined. The total absence of hematuria, proteinuria and leukocyturia, however, may be used to rule out heavy infections in community surveys. There was no difference in reagent strip scores between women with genital and urinary schistosomiasis as compared with those with urinary tract lesions alone. Thus urine analysis reagent strip readings do not help to discriminate between S. haematobium infected women with and without FGS.

Reversibility of lower reproductive tract abnormalities in women with Schistosoma haematobium infection after treatment with praziquantel--an interim report.

Little is known whether and to what extent antiparasitic treatment cures female genital schistosomiasis (FGS). Using a standard protocol, of twenty-one women with FGS nine were re-examined at two to nine weeks after they had been treated with praziquantel at a single dose of 40 mg/kg. Symptoms related to pathology of the urinary tract and to a lesser extent of genital pathology subsided in most patients. Schistosoma haematobium ova were no longer detectable in urine of any of the patients post-treatment. Efficiency of chemotherapy against adult worms was confirmed by the disappearance of circulating anodic antigen (CAA) in serum. Sandy patches showed resolution in two of four cases after chemotherapy. Papillomata due to schistosomiasis alone improved, but persisted in mixed infection with human papilloma virus (HPV) or when HPV was the only underlying cause. In one patient ulcera could not be related with certainty to schistosomiasis at admission, but resolved after treatment with parziquantel. Leukoplakia (two cases) was not influenced by chemotherapy, or even increased during follow-up, regardless of whether ova had been detected or not. Although the follow-up period was rather short, time intervals were not standardized, and a relatively small number of patients was investigated, it could be shown that genital pathology due to sequestered S. haematobium ova is, at least partially, reversible already two to nine weeks after killing the adult worms by praziquantel. This is paralleled by a normalization of inflammatory immune responses detectable in histological sections and vaginal lavage.

Combined use of urinary UGP and serum CA 125 in the diagnosis of gynecological cancers.

UGP, the beta-core fragment of human chorionic gonadotropin has been proposed as a tumor marker for gynecological malignancies. This fragment may be detected in a single morning-specimen of urine using an enzyme immunoassay. In this study, the diagnostic usefulness of urine UGP and serum CA 125 measurement for gynecological neoplasias (149 cases) was evaluated using a control group of patients with benign gynecological diseases (69 cases) and healthy females (99 cases). Considering the neoplastic patients in comparison to patients with benign diseases, the best diagnostic efficiency (78%) was found to correspond to a cut-off level of 120 pmol/mol creatinine the sensitivity being 73% and the specificity 90%. With this cut-off, an efficiency of 82% for healthy controls was obtained. Since the menopausal condition increases UGP levels, and though no significant difference for UGP was found between healthy subjects and patients with benign diseases, we decided to consider the reference populations as a single group. Thus, we evaluated the UGP performance on the basis of menopausal status. When a specificity of 95% was fixed, the cut-off values were 120 and 180 pmol/mol creatinine for pre- and postmenopausal women respectively, the sensitivity being 73% and 64%. Finally the combined evaluation of UGP and CA 125 improved their individual clinical efficiency for the diagnosis of ovarian serous cystadenocarcinomas, assuring a sensitivity of 86% and a specificity of 89%.

Beta-core fragment (beta-core/UGF/UGP), a tumor marker: a 7-year report.

In 1988 we published three papers describing immunoassay results for urine beta-core fragment as a marker of gynecological cancers. Many other papers have been published since, and three commercial immunoassays have been established. beta-Core fragment is called beta-core, UGF, or UGP by different commercial vendors. To avoid confusion we call it beta-core/UGF/UGP here. In this 7-year report, we compare the three commercial assays, establish cutoff limits, and use the Ciba-Corning kit for two large studies. The first was a retrospective study, measuring beta-core/UGF/UGP in gynecological cancer and control urines accumulated in our freezers (n = 486). The second is a first prospective study, testing over a 16-month period beta-core/UGF/UGP levels in urines of all new patients attending the Gynecology Oncology Clinic (n = 548). In the retrospective study, elevated beta-core/UGF/UGP levels ( > 1.9 ng/ml) were detected in 11% of urines from healthy individuals (n = 132), in 11% from women with benign gynecological disease (n = 196), in 44% from cervical cancer (n = 68), 56% from ovarian cancer (n = 54), and 47% from endometrial cancer (n = 38). Altogether, beta-core/UGF/UGP levels were elevated in 50% of 170 samples from gynecological cancers. Overall, sensitivity increased with advancing stage of malignancy. Sensitivity was 28% for stage I, 50% for stage II, 47% for stage III, and 68% for stage IV malignancies. In the prospective study very similar results were recorded. Elevated beta-core/UGF/UGP levels ( > 1.9 ng/ml) were detected in 11% of urines from healthy individuals (n = 99), 11% from individuals with benign gynecological disease (n = 196), 7% from women with carcinoma in situ (n = 28), in 42% of samples from cervical cancer (n = 69), 56% from ovarian cancer (n = 59), and 52% from endometrial cancer. Altogether, beta-core/UGF/UGP levels were elevated in 48% of 225 gynecological cancer samples. Overall, sensitivity increased with advancing stage of malignancy. Sensitivity was 29% for stage I, 66% for stage II, 60% for stage III, and 77% for stage IV malignancies. In both studies sensitivity for beta-core/UGF/UGP increased with advancing stage of disease. Sensitivity for cervical and endometrial cancers was slightly lower than that for ovarian malignancies. This difference may be due to the preponderance of advanced-stage-disease patients in the ovarian cancer group. beta-Core/UGF/UGP may be a general stage-dependent marker for all gynecological cancers. The same false-positive results and very similar sensitivity values were found in a retrospective and a prospective study. They confirm each other, and suggest a definitive false-positive rate and sensitivity of this tumor marker for gynecological cancers.

[Preliminary experience with a new tumor marker in obstetrics and gynecology: UGP (Urinary Gonadotropin Protein)]. / Esperienze preliminari con un nuovo marcatore tumorale in ostetricia e ginecologia: UGP.

AIM: To evaluate the use of UGP (urinary gonadotropin protein) as a tumor marker in gynaecologic and obstetric malignant diseases. MATERIALS AND METHODS: The study was carried out in the division of Gynaecology and Obstetrics of the Ospedali Riuniti in Bergamo. 63 patients, with obstetric or gynaecologic benign or malignant diseases, entered the study. 66 healthy volunteers were examined as a group-control. In both the groups UGP levels were determined in morning urine, using an immunoenzymatic commercial kit. RESULTS: Results, expressed in fmol UGP/ml of urin, show that UGP is produced by several neoplasms, but the false-positive percentage is still high; a higher precision can be obtained with an accurate choice of the cut-off value and with a standardization of the analytical technics. Besides, the contemporary determination of UGP and CA 125 levels reduces the possibility of false-positive and false-negative results. CONCLUSIONS: More studies must be carried out to confirm the value of UGP as a tumor marker in obstetrics and gynaecology. Anyway, this recently purified protein can already be useful, in combination with the usual tumor markers, in the prompt diagnosis and management of primary neoplasms or recurrences, with a higher sensibility in comparison with traditional clinical and radiological examinations.

Urinary gonadotropin peptide: collection of specimens and cutoff levels.

A low-molecular-weight form of human chorionic gonadotropin (hCG), urinary gonadotropin peptide (UGP), has been isolated from the urine of pregnant women and of patients with cancer, mainly of gynecological origin. The clinical value of UGP measurement in gynecological diseases is under investigation but a preliminary study is necessary in order to ascertain whether there is a circadian rhythm in UGP production, to clarify the best way to express the results, and to establish the cutoff and decisional values. In our work we demonstrated a significant correlation between the UGP output and the UGP excretion normalized for urinary creatinine. A very significant agreement was even found in 24-hr urine collections and UGP concentration of a single morning specimen from the same patients. No evident circadian rhythm was found, although some patients presented morning levels of UGP higher than in other collections. UGP postmenopausal levels were higher than premenopausal. The cutoff level, adopting the 95.0 percentile, was 200 pmol/mol creatinine.

Urinary gonadotropin peptide (UGP) as a marker of gynecologic malignancies.

Urinary gonadotropin peptide (UGP) was measured in 866 urines from normal women and women with benign and malignant gynecologic disease using the Triton UGP enzyme immunoassay. The greatest level of overexpression of the marker was observed in patients with ovarian cancer. Using a cutoff of 4 fmol/mg creatinine, UGP was overexpressed in samples from 2% of normal premenopausal women, 15% of normal postmenopausal women, 5% of women with benign gynecologic disease, and 59% of women with ovarian cancer. UGP expression was independent of the histologic type of ovarian cancer. The expression of UGP and CA 125 were not correlated and use of the two markers in tandem increased the sensitivity of detection of disease by greater than 20% over that which was observed using each marker individually. UGP levels were correlated with clinical status, and doubled in value in 67% of patients with progressive disease, and were halved in 93% of patients who were in remission at the time of the study.