Glycosaminoglycan and transforming growth factorb1 changes in human plasma and urine during themenstrual cycle, in vitro fertilization treatment, andpregnancyPierina De Muro, M.D.,a Giampiero Capobianco, M.D., Ph.D.,b Marilena Formato, M.D.,a

Antonio Junior Lepedda, M.D.,a Gian Mario Cherchi, M.D.,a Laila Gordini, M.D.,b

and Salvatore Dessole, M.D.b

a Department of Physiological, Biochemical and Cellular Sciences, University of Sassari, Sassari, Italy; and b Department ofPharmacology, Gynecology and Obstetrics University of Sassari, Sassari, Italy

Objective: To evaluate transforming growth factor b1 (TGF-b1) and glycosaminoglycans (GAG) changes in hu-man plasma and urine during the menstrual cycle, IVF-ET, and pregnancy.Design: Prospective clinical study.Setting: University hospital.Patient(s): Thirteen women with apparently normal menstrual cycle (group 1); 18 women undergoing IVF-ET(group 2); and 14 low-risk pregnant women (group 3).Intervention(s): We assayed plasma and urine concentrations of TGF-b1, urine content, and distribution of GAG.Blood and urine samples were collected during days 2 to 3, 12 to 13, and 23 to 24 in group 1; in group 2, sampleswere obtained at menstrual phase, oocyte pick-up day, and 15 days after ET; in group 3, samples were obtainedduring gestational weeks 10–12, 22–24, and 30–32 and 1 month after delivery.Main Outcome Measure(s): Changes in TGF-b1 and GAG content.Result(s): The mean value of total urinary trypsin inhibitor/chondroitin sulfate (UTI/CS) showed a distinct peak atday 12 of the menstrual cycle in the fertile women in whom we monitored the ovulatory period. In the IVF-ETgroup, GAG distribution and TGF-b1 levels showed significant differences during the cycle. We observed in-creased levels of plasma TGF-b1 15 days after ET. A significant increase of total UTI/CS value with increasinggestation was detected.Conclusion(s): Transforming growth factor b1 and GAG levels could represent an additional tool to monitor re-productive events and could be useful, noninvasive markers of ovulation and ongoing pregnancy. (Fertil Steril!

2009;92:320–7. "2009 by American Society for Reproductive Medicine.)

Key Words: Transforming growth factor b1, glycosaminoglycans, urinary trypsin inhibitor, women

Various female reproductive processes, such as ovulation,fertilization, implantation, and pregnancy, are inflamma-tory-like responses in which proinflammatory and anti-inflammatory cytokines are involved.

GAG are a group of complex anionic heteropolysacchar-ides that in tissues, with the exception of hyaluronic acid,are linked to core protein-forming proteoglycans (PG) andparticipate in processes including resistance to compressiveforce and tissue hydration and in a variety of cell communi-cation events. Recent data have shown that GAG initiate andcontrol events associated with inflammation (1). Glycosami-noglycans influence cytokine/chemokine production, leuko-cyte recruitment, and inflammatory cell maturation (2–4).

Low-sulphated chondroitin sulphate (LSC) is the GAGchain of the LSC-proteoglycan (LSC-PG).

Urinary trypsin inhibitor (UTI), also defined as LSC-PG,a Kunitz-type serine protease inhibitor, occurs in blood andurine as the major chondroitin sulphate PG (5). It is an acidicglycoprotein composed of a core protein of 143 amino acidsand two carbohydrate side chains: an oligosaccharide anda GAG chain consisting of a relatively low-sulphated chon-droitin sulphate. In urine, its concentration is very low innormal conditions and increases in inflammatory diseases,malignant diseases, pregnancy, and postsurgical states (6,7) and after the administration of steroids (6, 8). Urinarytrypsin inhibitor is the light chain of inter-a-trypsin inhibitorand pre-a-trypsin inhibitor, which are assumed to be precur-sors of UTI. During the acute-phase response, elastasemetabolizes pre-a-trypsin inhibitor and inter-a-trypsin inhib-itor to liberate UTI (9). Because UTI is quickly excreted intourine, its urinary excretion has been regarded as an index ofsystemic UTI concentration. Moreover, this glycoprotein dis-plays functions related to the reproductive system: inter-a-trypsin inhibitor (including UTI as a component) binds

Received February 2, 2008; revised May 2, 2008; accepted May 19, 2008;published online August 11, 2008.

P.D.M. has nothing to disclose. G.C. has nothing to disclose. M.F. hasnothing to disclose. A.J.L. has nothing to disclose. G.M.C. has nothingto disclose. L.G. has nothing to disclose. S.D. has nothing to disclose.

Reprint requests: Salvatore Dessole, M.D., Department of Pharmacology,Gynecology and Obstetrics, University of Sassari, Viale San Pietro 12,Sassari 07100, Italy (FAX: 39-079-228265; E-mail: dessole@uniss.it).

Fertility and Sterility# Vol. 92, No. 1, July 2009 0015-0282/09/$36.00Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2008.05.066

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hyaluronic acid and stabilizes the extracellular matrix of themouse cumulus–oocyte complex (10), which is essential foroocyte maturation and ovulation. Furthermore, UTI helpsprevent preterm delivery by inhibiting the contraction ofthe uterus (11).

In this inflammation scenario, transforming growth factorb1 (TGF-b1) plays an important role as a potent modulatorof immune and anti-inflammatory responses. This pleiotropiccytokine is associated with tissue remodeling events andreproductive processes because of its powerful effects on cel-lular proliferation and differentiation, angiogenesis, andimmunomodulation. It has been previously shown that it isproduced in both granulosa and theca cells (12), and it mightbe involved in follicular growth. Moreover, this immunomo-dulating cytokine has also been detected in human endome-trium at the time of implantation and might suppressinappropriate responses at the fetal–maternal interface (13).

To investigate the relationships between reproductivestates and TGF-b1, UTI, and the other urinary GAG species,we examined their plasma and urine content in conditions inwhich the steroid levels are increased (during the regularmenstrual cycle, during IVF-ET procedures, and during preg-nancy) to verify their possible use as markers of ovulationand of abnormal conditions in pregnancy.

MATERIALS AND METHODSSubjectsThe study population consisted of 45 women, divided intothree groups. Group 1 comprised 13 with apparently normalmenstrual cycle (aged 27.4 ! 6.8 years [mean SD]; range,20–40 years); group 2 comprised 18 women who underwentovarian hyperstimulation with IVF-ET (aged 36.4 ! 4.0years; range, 30–41 years); and group 3 comprised 14 womenwith low-risk pregnancy who delivered healthy infants atterm (aged 31.3 4.1 years; range, 28–41 years). Ten healthy,nonpregnant women (aged 30.2 ! 3.7 years; range, 24–40years) served as controls.

Informed consent was obtained from all women. Institu-tional review board approval was obtained.

Peripheral blood and early morning urine were collectedfrom every woman for three periods: for fertile women atthe menstrual phase (days 2 to 3), ovulatory phase (days 12to 13), and luteal phase (days 23 to 24); and in the IVF-ETgroup at the menstrual phase (days 2 to 3), oocyte pick-upday, and 15 days after ET. In pregnant women these collec-tions were made at weeks 10–12, 22–24, and 30–32 and 1month after delivery. In the control group blood and urinesamples were collected independently ofmenstrual cycle day.

Moreover, 8 of the 13 volunteer fertile women collectedearly-morning urine samples for each day of the menstrualcycle from day 8 to day 17.

No women with proteinuria (cut-off, 30 mg/dL) wereincluded in this study. Other exclusion criteria were hyperten-sion (blood pressure R140/90 mm Hg) and the presence of

active infectious disease or medical complications, includingautoimmune disorders, endometriosis, diabetes mellitus, in-flammatory conditions, and tobacco use; for fertile women,hormonal therapy as well as symptoms of urinary infectionjust before or during the menstrual cycle under study; for preg-nant women we excluded those with multiple pregnancies.

The inclusion criteria for the IVF-ET group were infertility>3 years, basal serum FSH level <10 IU/L, and normal PRLlevels. The indications for IVF-ET treatment included tubal(n " 3), male (n " 8), and unexplained (n " 7) factors ofinfertility. All patients were treated with the following proto-col for ovarian stimulation. The cycle was started by initialsuppression of ovarian function with the GnRH analogue trip-torelin (Decapeptyl; Ipsen, Milan, Italy) on day 23 of the men-strual cycle. After down-regulation was verified, at days 2 to 3of the subsequent menstrual cycle, daily SC injections of 150–225 IU of recombinant FSH (Gonal F; Serono, Rome, Italy)were given. This was continued until the leading folliclereached a diameter of R18 mm and the serum level of17b-E2 reached 750 ng/mL. Then 10,000 IU of hCG (Gonasi;AMSA, Rome, Italy) was given IM. Thirty-six hours later, oo-cyte pick-up was performed by ultrasound-guided transvagi-nal follicle aspiration, and 2 to 3 days later ETwas carried out.

Isolation and Analysis of Urinary GAG and PGEarly-morning urine samples were collected. Standardurinalysis was performed on each urine specimen to screenfor the presence of urinary tract infection, proteinuria, orhematuria.

GAG were isolated as described elsewhere (14). Glycos-aminoglycan composition was determined, after solubiliza-tion with water, by electrophoresis on cellulose acetatestrips in a discontinuous buffer. GAG identification was per-formed by comparison with co-migrating standard GAG andUTI (SCIPAC, Sittingbourne, United Kingdom) and by treat-ing aliquots of the samples at 37#C for 18 hours before elec-trophoresis with specific chondro/dermato-lyases and, insome cases, with papain. Glycosaminoglycan levels were ex-pressed as relative percentages by densitometric analysis ofAlcian blue-stained strips quantitated using computer-baseddigital image processing (Thunder Scan; Biosoft, Cam-bridge, United Kingdom).

Structural Analysis of Urinary PGPurification of PG Urine samples (150mL) fromwomen pos-itive for LSC-PG were processed using the same procedurebut with a discontinuous salt gradient elution (0.35 mol/L,0.45 mol/L, 0.55 mol/L, and 1.2 mol/L LiCl in 0.02 mol/LTris-HCl, pH 8.6).

The chromatographic fraction eluted with 0.35 mol/L LiClwas precipitated in absolute ethanol, centrifuged at 10000 $g for 30 minutes, and dried at 60#C. After reconstitution with100 mmol/L ammonium acetate, pH 8.0, the digestion withchondroitin ABC and AC lyases was performed at 37#C for

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3 hours (15). The digestion mixture was boiled to inactivatethe enzyme and centrifuged. The pellet was analyzed forprotein composition by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE), and the supernatantwas dried at 60#C, solubilized with distilled water, and usedfor disaccharide characterization by capillary zone electro-phoresis (CZE) analysis.

SDS-PAGE Analysis The electrophoretic analysis of theprotein core was performed in reducing conditions (in thepresence of 3% mercaptoethanol) on precast 4%–12% poly-acrylamide Tris-glycine gels (Invitrogen, Carlsbad, CA) byusing a Novex system (Invitrogen) for 1.5 hours at constantvoltage (150 V) at 22#C. After electrophoresis the gelswere stained with Simply Blue SafeStain (Invitrogen).

CZE Analysis of Unsaturated Disaccharides Analysis wasperformed essentially as described by al-Hakim (16) in a P/ACE capillary electrophoresis system (Beckman Instruments,Fullerton, CA) applying 20 nL of sample under nitrogen pres-sure (0.5 psi for 3 seconds) in a 50mm internal diameter and 57cm long (40 cm to the detection window) uncoated fused-sil-ica capillary, using a 75-mmol/L borate buffer, pH 10.4. Theseparating conditions (15 kV) were reached in 20 secondsand held at a constant voltage for 25 minutes. Separationswere carried out at 28#C and monitored by ultraviolet detec-tion at 232 nm. Standard disaccharides 2-acetamido-2-de-oxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)-D-galactose(Ddi-0scs), 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyra-nosyluronic acid)-4-O-sulfo-D-galactose (Ddimono4S), and2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronicacid)-6-O-sulfo-D-galactose (Ddi-mono6S) were purchasedfrom Seikagaku (Cod. 400570; Tokyo, Japan).

Analysis of TGF-b1Urine samples were centrifuged at 3000 $ g for 10 minutes,and clear supernatants were stored at %20#C.

The plasma and urine concentrations of TGF-b1 (activeplus acid-activatable latent) were measured by ELISA usingkits from Promega (Madison, WI). The minimum detectableconcentration of TGF-b1 by the assay was 32 pg/mL. The in-tra-assay coefficient of variance (CV) was<4.5% and the in-terassay CV <19.1%. To activate latent TGF-b1 the sampleswere incubated with 1 N HCl for 1 hour at room temperature.Then they were titrated with 1 N NaOH to pH 7.6.

Assessment of Serum Hormonal ProfileSerum levels of LH, FSH, E2, and P were measured byenzyme-immunoassay methods at the reproductive endocrineunit laboratory of the Department of Pharmacology, Gyne-cology and Obstetrics, University of Sassari, Italy.

Other Analytic MethodsUrinary creatininewasmeasured by the Jaff!e method (SentinelCH, Milan, Italy). Urinary protein content was measured withurine reagent strips (Menarini Diagnostics, Florence, Italy).

Statistical AnalysisValues are reported asmean! SD andmedians with ranges. In-tragroup differences were tested using one-way repeated-mea-sures analysis of variance on ranks, and intergroup changesusing one-way analysis of variance on ranks when data failedthe normality test. The significance level was defined asP<.05. Comparison between frequencies was made by the c2

test.

RESULTSTotal urinary GAG levels, expressed as milligrams hexuro-nate per gram of creatinine, did not present statistically sig-nificant intragroup differences among the three categoriesof subjects examined, but they were significantly higher inthe urine of pregnant women at the second and third trimesterof gestation compared with nonpregnant women (6.05! 2.69and 6.29 ! 1.96 vs. 3.89 ! 1.35; P<.05).

As shown in Figure 1, in all the examined women we wereable to detect two major bands: chondroitin sulfate (CS) andheparan sulphate (HS). However, many women showedanother component with slower electrophoretic migrationthan HS that was identified as a GAG–protein complex(LSC-PG or UTI) (17). In some urine samples we foundtwo LSC bands with different electrophoretic migration,which disappeared after separate treatments with either chon-droitinase AC and ABC. The band slower than CS is referred

FIGURE 1

Electrophoretic profiles of urine GAG. (A) Theelectrophoretic component, slower than UTI,referred to as UTI 2, was detected in some women’surine samples (lanes 1 and 2). (B) Electrophoreticpatterns of urine GAG observed in a fertile womanduring the ovulatory period of the menstrual cycle(lanes 3–6) from days 10 to 13.

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322 De Muro et al. Glycosaminoglycans and TGF-b1 in women Vol. 92, No. 1, July 2009

FIGURE 2

Structural analysis of LSC-PG. (A) SDS-PAGE patterns of UTI standard (lane 3) and urine-purified LSC-PG aftertreatment with chondroitin ABC and AC lyases (lane 2). (B) Typical electropherogram of standard D-disaccharides (0.3 pmol each). (C)D-disaccharides obtained from urine-purified LSC-PG upon depolymerizationby chondroitin ABC/AC lyases.

De Muro. Glycosaminoglycans and TGF-b1 in women. Fertil Steril 2009.

TABLE 1Urinary distribution of UTI and UTI derivatives in the examined women.

Patient group n UTI UTI 2 SM-LSC LSC

Fertile women (days 2 to 3) 13 5/13 0/13 0/13 0/13Fertile women (days 12 to 13) 13 5/13 4/13 9/13a 3/13Fertile women (days 23 to 24) 13 6/13 2/13 1/13 0/13IVF-ET women (days 2 to 3) 18 14/18 1/18 0/18 0/18IVF-ET women (oocyte pick-up day) 18 14/18 1/18 1/18 3/18IVF-ET women (15 days after ET) 11 11/11 1/11 2/11 0/11Pregnant women (weeks 10–12) 14 11/14 2/14 3/14 2/14Pregnant women (weeks 22–24) 14 13/14 4/14 8/14 1/14Pregnant women (weeks 30–32) 14 14/14 4/14 13/14b 0/14Postpartum women (1 mo after delivery) 8 3/8 0/8 2/8 2/8

a c2 " 10.9, P" .0010 vs. fertile women (days 2 to 3) and c2 " 7.96, P" .0048 vs. fertile women (days 23 to 24).b c2 " 11.8, P" .0006 vs. pregnant women (weeks 10–12).

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to as LSC and that with a slower electrophoretic migrationthan LSC as slow migrating-LSC (SM-LSC).

Sometimes an additional band, slower than UTI, wasdetected. This component, which completely disappeared only

when papain treatment preceded chondroitinase AC and ABCdepolymerization, is hereafter referred to as UTI 2 (Fig. 1A).

For structural analysis, LSC-PG was depolymerized withchondroitin ABC and AC lyases. Afterward its protein corewas analyzed by SDS-PAGE and its unsaturated disaccharidecomposition was determined by high-performance CZE. TheLSC-PG contained mostly 30-kDe material, although lower-molecular-weight bands and traces of approximately 63-kDematerials were also found (Fig. 2).

A typical CZE separation of standard and urinary LSC-derived D-disaccharides is presented in Figure 2. Resultsshowed that LSC-PG is composed of approximately 68%non-sulphated disaccharides and 27% 4-sulphated disaccha-rides, with trace amounts of 6-sulphated disaccharides. Theobtained composition of urinary LSC-derived D-disaccha-rides is in complete accordance with those previously re-ported for UTI (18).

Table 1 shows the distributions of UTI and UTI derivativesin the three groups of subjects.

We observed the urinary excretion of UTI 2 and SM-LSC atthe ovulatory and luteal phases in the fertile women, witha higher frequency of SM-LSCwith respect to the other phases,and the progressively increased excretion of UTI derivatives,chiefly SM-LSC, during pregnancy. In fact, in late pregnancy,SM-LSC frequency was higher than in the initial stage.

All subjects of group 1 showed serumhormonal patterns in-dicating normal menstrual cycles. Estradiol levels (mean !SD) for the various phases of the menstrual cycle were asfollows: 24.35 ! 19.43 pg/mL (menstrual phase), 100.12 !50.20 pg/mL (periovulatory phase), and 102.84 ! 26.76 pg/mL (luteal phase). Progesterone levels were 0.63 ! 0.53 ng/mL (menstrual phase), 0.76 ! 0.54 ng/mL (periovulatoryphase), and 12.47 ! 2.02 ng/mL (luteal phase). All luteal-phase P levels were in the ovulatory range (PR10 ng/mL).

To verify whether the ovulatory process affects urinaryGAG and TGF-b1 metabolism, we monitored the preovula-tory and ovulatory period (from days 8 to 17) in 8 of the 13fertile women, observing a modification of the GAG distribu-tion from day 11 to 15, with characteristic patterns in threewomen. Figure 1B depicts this urinary profile in one of thesethree women.

Because of the heterogeneity of UTI derivatives, the distri-bution of GAG is expressed as total UTI/CS and HS/CSratios, where total UTI is the sum of UTI plus UTI 2, LSC,and SM-LSC.

When mean levels at each day of the preovulatory andovulatory periods were compared, significant intragroup dif-ferences were detected only for UTI/CS ratio (day-12 levelshigher than other days except for day 13; Fig. 3A and B).These results imply a modification of GAG excretion in a nar-row time interval.

By analyzing the amounts of GAG and TGF-b1 in the fer-tile women at the three time points of the menstrual cycle,

FIGURE 3

Total concentration and relative content of urinaryGAG and TGF-b1 in the examined women. (A) Meanvalues of total GAG content (blue circles), expressedas milligrams hexuronate per gram creatinine, andrelative contents estimated as total UTI/CS (pinksquares) and HS/CS (yellow triangles) ratios,detected in 8 fertile women during the periovulatoryand ovulatory periods of the menstrual cycle. (B)Mean TGF-b1 levels (blue squares) detected in thesame subjects. (C) Total UTI/CS (pink squares) andSM-LSC/CS (blue circles) values and E2 levels(yellow triangles), expressed as picograms permilliliter, determined in one IVF-ET patient from day 4of pregnancy until 1 month after birth.

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324 De Muro et al. Glycosaminoglycans and TGF-b1 in women Vol. 92, No. 1, July 2009

significant differences were found between the ovulatory,menstrual, and luteal phases, as depicted in Table 2.

In the IVF-ET group, the median value of HS/CS ratiowas significantly increased at oocyte pick-up day with re-spect to the menstrual phase (Table 3), owing to a signifi-cant decrease of CS relative content. Conversely, themedian value of total UTI/CS ratio did not change signifi-cantly during the stimulated cycles but tended to be higher15 days after ET; this was also true of plasma TGF-b1levels, for which the difference was statistically significant(phase 3 vs. phases 1 and 2, P<.05). In 3 of the 18 womenwho underwent IVF-ET the treatment produced successfulovarian hyperstimulation, and in 1 of these patients we de-termined the total UTI/CS, SM-LSC/CS values, and 17b-E2

levels from day 4 of pregnancy until 1 month after birth(Fig. 3C).

In pregnancy, significant differences were detected forGAG ratios and TGF-b1 levels between the third trimesterand the other phases (Table 4). The modification in GAG dis-tribution was due to the increased urinary excretion of SM-LSC and to the significant decrease in CS relative contentin the third compared with the second and first trimesters(Table 5).

DISCUSSIONIn the present study the qualitative and quantitative evalua-tion of both GAG and PG allowed us to detect complex elec-trophoretic patterns in the examined groups.

Interestingly, the main heterogeneity was related to theUTI derivatives (UTI 2, LSC, and SM-LSC). Urinary excre-tion of UTI 2 and SM-LSC were only detected in the ovula-tory and luteal phases of the menstrual cycle, and SM-LSCincreased during gestation. It is possible that UTI 2 representsa product of degradation of inter-a-trypsin inhibitor, in whicha fragment of either heavy chain (H1 or H2) is preserved.

The total UTI/CS mean value showed a distinct peak at day12 of the menstrual cycle in the fertile women in whom wemonitored the periovulatory period. Moreover, in 3 of these8 women we detected a 1-day modification of the GAG elec-trophoretic pattern, with the urinary excretion of UTI deriva-tives and a decreased excretion of CS. By evaluating thewhole cycle, only UTI/CS ratio showed a significant variationat the ovulatory phase compared with the menstrual and lu-teal phases. This agrees with the finding that in a group of fer-tile women with anovulatory cycle (P levels <10 ng/mL atluteal phase) no statistical differences in GAG distributionwere detected (data not shown).

TABLE 2Glycosaminoglycans and TGF-b1 levels in fertile women.

VariableMenstrual phase

(days 2 to 3)Ovulatory phase(days 12 to 13)

Luteal phase(days 23 to 24)

Total UTI/CS 0.42 (0–1.03) 1.00 (0–3.60)a 0.23 (0–1.50)HS/CS 0.95 (0.50–2.20) 1.49 (0.66–3.16)a 0.87 (0.43–1.42)Plasma TGF-b1 (ng/mL) 20.70 (0.54–32.70) 22.30 (1.10–38.40) 32.70 (12.00–88.00)b

Urine TGF-b1 (ng/g creatinine) 1.60 (0–10.00) 4.00 (0–57.70) 0 (0–10.00)

Note: Data reported as median (range). Total UTI is the sum of UTI plus UTI 2, LSC, and SM-LSC.a P< .05 vs. menstrual and luteal phase.b P< .05 vs. menstrual and ovulatory phase.

De Muro. Glycosaminoglycans and TGF-b1 in women. Fertil Steril 2009.

TABLE 3Glycosaminoglycans and TGF-b1 levels in IVF-ET group.

Variable Phase 1 Phase 2 Phase 3

n 18 18 11Total UTI/CS ratio 0.72 (0–2.50) 1.05 (0–2.10) 1.04 (0.60–2.30)HS/CS ratio 0.83 (0.52–1.39) 1.00 (0.58–2.34)a 0.92 (0.59–1.31)Plasma TGF-b1 (ng/mL) 29.00 (0–105.0) 39.90 (0–120.0) 95.70 (1.00–202.5)b

Urine TGF-b1 (ng/g creatinine) 0 (0–2.00) 0 (0–34.30) 0 (0–10.00)

Note: Data reported as median (range). Phase 1" days 2 to 3, phase 2" oocyte pick-up day, phase 3" 15 days after ET.a P< .05 vs. phase 1.b P< .05 vs. phases 1 and 2.

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Recently we observed a heterogeneous electrophoretic pat-tern of UTI derivatives (17), with the presence of LSC orSM-LSC in the urine of nephropathic patients. It is possiblethat in pathologic conditions and in physiologic states suchas reproductive processes an excretion of UTI fragments afterinflammatory-inducedproteolysis occurs, and that their hetero-geneity is related to the severity of the inflammatory process.

An involvement of UTI in the later stage of pregnancy hasbeen reported. In fact, administration of UTI prevents pre-term delivery by inhibiting both cervical ripening (19) anduterine smooth muscle contraction (20).

In the present study we observed a significant increase oftotal UTI/CS ratio with increasing gestation, and it was inpart attributable to the increased urinary excretion of SM-LSC and to the significant relative decrease of CS, in partic-ular in late pregnancy. This reduction of CS relative contentduring pregnancy is in agreement with the results of Weilkeet al. (21), who observed an increase in the activity of serumxylosyltransferase (the chain-initiating enzyme in the biosyn-thesis of CS), up to 5 weeks after conception, followed by

a decline in the next weeks of gestation. High E2 concentra-tions during pregnancy and the menstrual cycle lead to in-creased water content in the connective tissue. This seemsto be caused by an increased synthesis of polyanionic CS-PG, which are highly hydrated.

Moreover, in the present study we also detected the urinaryexcretion of SM-LSC in the first trimester of pregnancy ina patient who progressed to a preterm delivery. The mecha-nism by which uterine smooth muscle switches from a quies-cent state during pregnancy to contraction at labor has beenthe subject of investigation for years. Parturition occursthrough the up-regulation of a group of pro-labor genes orthrough the down-regulation of a group of genes such as thosefor nitric oxide synthase and UTI (22), which may decreaseuterine contractility and maintain the uterus in a state of qui-escence during pregnancy.

With regard to TGF-b1 levels, we observed increasedplasma levels 15 days after ET, probably related to the ovar-ian hyperstimulation. This is consistent with the results ofFried et al. (23), who reported follicular expression of

TABLE 4Urinary GAG and plasma and urine TGF-b1 levels in pregnancy.

Variable Phase 1 Phase 2 Phase 3 Phase 4 Controls

n 14 14 14 8 10Total UTI/CS 1.07 (0–2.00) 2.00 (0–4.40)a 2.70 (0–17.00)b 0.48 (0–3.05) 0 (0–1.40)HS/CS 1.05 (0.60–2.33) 1.24 (0.90–2.80)a 1.34 (1.10–6.90)a 1.09 (0.43–1.84) 0.91 (0.43–1.60)Plasma

TGF-b1 (ng/mL)10.00 (0–57.30) 22.50 (7.00–43.00) 56.80 (4.30–170.0)c 20.00 (9.00–79.30) 18.00 (0–14.00)

Urine TGF-b1(ng/g creatinine)

0 (0–3.80) 1.50 (0–3.00) 14.50 (9.00–44.00)b 0 (0–5.00) 0.90 (0–10.00)

Note: Pregnant women: phase 1 " weeks 10–12, phase 2 " weeks 22–24, phase 3 " weeks 30–32, phase 4 " 1 monthafter delivery.

a P< .05 vs. controls.b P< .05 vs. phase 1, phase 2, phase 4, and nonpregnant women (controls).c P< .05 vs. phase 1, phase 2, and phase 4.

De Muro. Glycosaminoglycans and TGF-b1 in women. Fertil Steril 2009.

TABLE 5Relative content of urinary GAG in pregnancy.

Phase n UTI D UTI 2 D LSC (%) SM-LSC (%) HS (%) CS (%)

1 14 27.20 ! 15.79 4.00 ! 8.46 36.81 ! 13.01 30.18 ! 8.42a

2 14 30.41 ! 13.89 11.60 ! 11.79b 33.50 ! 11.74 25.10 ! 6.51c

3 14 30.90 ! 12.60 21.78 ! 9.52d 27.61 ! 4.36 18.04 ! 6.94d

4 8 26.68 ! 13.71 4.86 ! 9.45 33.68 ! 5.09 40.71 ! 21.52

Note:Data presented asmean!SD. Pregnant women: phase 1"weeks 10–12, phase 2"weeks 22–24, phase 3"weeks30–32, phase 4 " 1 month after delivery.

a P< .05 vs. phase 4.b P< .05 vs. phase 1.c P< .05 vs. phase 1 and phase 4.d P< .05 vs. phase 1, phase 2, and phase 4.

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TGF-b1 differentially regulated by FSH and LH: it is stimu-lated by FSH and inhibited by LH.

Successful pregnancy depends on the release and action ofan array of cytokines and other immunomodulators by cellsof the fetoplacental unit. Trophoblasts are important in thiscontext, producing a number of cytokines, including bothproinflammatory and anti-inflammatory factors (24). Thispresumes a cytokine balance, in which the actions of locallyproduced proinflammatory cytokines (Th1) are regulated byanti-inflammatory factors (Th2) within the placenta in nor-mal gestation.

It is possible that the increased levels we observed in latepregnancy are related to regulatory role of TGF-b1 ontrophoblast invasion and proliferation on the basis of the dem-onstration that peak expression of messenger RNA encodingTGF-b1 in human placenta occurred at weeks 17 and 34 ofgestation, time points coincident with the cessation of tropho-blast invasion and cessation of absolute trophoblast growth,respectively (25). In addition, TGF-b1 has a role in excitabilityand contractility of the laboring uterus in late gestation (26).

In conclusion, to our knowledge, this is the first studyshowing the urinary excretion of UTI and UTI derivatives(chiefly SM-LSC) during the ovulatory phase in the normalmenstrual cycle, and progressively increased during preg-nancy. Our results provide additional evidence that ovulationand parturition may be likened to an inflammatory reaction,in which inflammation-induced proteolytic products of theplasma serine proteinase inhibitors are excreted in urine.

Thus the levels and distribution of GAG and PG, as well asthe UTI/CS ratio, could represent useful, noninvasivemarkers of ovulation and of a normally ongoing pregnancy.Studies are in progress to improve the analytic method sothat it enables a more simple and rapid determination of thesebiomolecules as a tool for large clinical investigations.

Further studies are needed to clarify their role as pretermlabor risk markers and to evaluate whether TGF-b1 levelsmight be an additional marker of alteration in the formationof the uteroplacental unit.

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