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Arch Gynecol ObstetDOI 10.1007/s00404-014-3179-7

MAternAl-FetAl MeDIcIne

The assessment of the relationship between amniotic fluid matrix metalloproteinase‑9 and zinc levels with adverse obstetric outcomes

Özkan Özdamar · Ismet Gün · Ercüment Müngen · Vedat Atay

received: 15 January 2013 / Accepted: 10 February 2014 © Springer-Verlag Berlin Heidelberg 2014

Conclusion We found that the amniotic fluid MMP-9 levels of patients who delivered preterm were significantly lower between the 16th and 19th gestational weeks.

Keywords Adverse obstetric outcomes · MMP-9 · Zinc · Preterm birth · Amniocentesis

Introduction

In recent decades, many diseases and pathological pro-cesses have found their physiopathological roots at the cellular and molecular levels. A large family of endopro-teinases known as matrix metalloproteinases (MMPs) has a primary role in the degrading and remodeling of extra-cellular matrix (ecM). MMPs are characterized by the presence of a catalytic zinc atom in their active center [1]. numerous reports have shown MMPs to play important roles in a variety of birth-related physiological processes, including cervical ripening, term labor, parturition, mem-brane rupture and placental detachment, in addition to some pathological situations, such as preterm birth, pre-term premature rupture of membranes (PPrOM) and preeclampsia [2–4].

Increased activity and amount of MMP-9 and other MMPs in fetal membranes, decidua, and amniotic fluid have been found to be associated with both term and preterm birth [4–6]. Similarly, increased amniotic fluid MMP-9 levels are related to the preterm premature rup-ture of membranes [7]. In some studies of preeclamptic women, MMP levels were reported to be elevated in the amniotic fluid and tissue-specimens obtained from these patients [8], while other studies identified decreased levels [9, 10]. Fetal growth restriction (FGr), an important rea-son of perinatal mortality and morbidity due to iatrogenic

Abstract Objective to examine the relationship between the amni-otic fluid MMP-9 and zinc levels during 16–19th gesta-tional weeks and perinatal outcomes.Method One hundred and seventeen singleton pregnan-cies that underwent genetic amniocentesis from Janu-ary 2005 through november 2009 were evaluated. Sub-jects were divided into two main groups: a control group (group 1) (n: 74), and an adverse obstetric outcomes group (group 2) (n: 43). Group 2 consisted of the fol-lowing: preterm birth group, gestational hypertension and preeclampsia group, gestational diabetes group, fetal growth restriction group, macrosomia group, and preg-nancy loss group. MMP-9 and zinc (Zn) values in the amniocentesis materials sampled between the 16th and 19th gestational weeks were analyzed retrospectively in terms of perinatal outcomes. Any significant difference among the groups was assessed by unpaired samples t test and the Mann–Whitney U test. Statistical significance was defined as p < 0.05.Results A comparison among groups showed no signifi-cant difference in terms of Zn results between the group 1 and 2 (p = 0.879). MMP-9 levels were significantly lower in both the preterm birth group (p = 0.043) and group 1 (p = 0.015).

this study was presented as an oral presentation in the 10th national Gynecologic and Obstetrics congress at 9–13 May 2012 at Maritim Pıne Beach Otel, Antalya, türkiye. It received the award for best abstract in the category of oral presentation.

Ö. Özdamar · I. Gün (*) · e. Müngen · V. Atay Department of Obstetrics and Gynecology, GAtA Haydarpasa training Hospital, Istanbul 34668, turkeye-mail: drsmetgun@yahoo.com; drismetgun@gmail.com

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prematurity, has been related to reduced concentrations of MMP-9 in placenta and tissue samples [11, 12]. In gesta-tional diabetes (GDM) cases, increased MMP-9 levels in serum, uterus and placental tissues have been shown [13, 14]. However, all these studies have been made in patients with PPrOM in the third trimester or active laboring patients, or with samples taken after pregnancy complica-tions, such as preeclampsia or gestational diabetes, have occurred. Small numbers of studies investigate the rela-tionship between perinatal outcomes and MMP levels in the early weeks of pregnancy, such as the 15–20th weeks. Between these weeks, a study made by Vadillo-Ortega et al. [7] measured the MMP-9 levels in amniotic fluid between the 12th and 16th gestational weeks, but the peri-natal outcomes of their cases have not been investigated. In our study, the relationship between the amniotic fluid MMP-9 and zinc levels in the 16–19th weeks of gestation and perinatal outcomes has been investigated. lavee et al. [15] evaluated the levels of MMP-2 in second trimester amniotic fluid of women with hypertensive disorders com-pared to normotensive women.

the objective of this study was to determine the value of amniotic fluid MMP-9 and Zn in the prediction of adverse obstetric outcomes in asymptomatic pregnancies that underwent genetic amniocentesis. Although it has been suggested by previous studies that preterm birth was cor-related with higher levels of amniotic fluid MMP-9 levels, we assessed whether the same correlation does exist in the earlier gestational weeks.

Materials and methods

Study design

ethics committee approval was obtained before the initia-tion of this study. this retrospective cross-sectional study was conducted in the period of January 2005–novem-ber 2009 at the Gülhane Military Medical Academy, Haydarpasa training Hospital, Obstetrics and Gynecology Service, Istanbul.

Of 239 pregnant women who underwent genetic amni-ocentesis between the 16th and 19th gestational weeks, 117 pregnancies that met the inclusion criteria and whose parturition records and data could be obtained were evalu-ated. Inclusion criteria for the study included singleton pregnancy, normal pregnancy course prior to the proce-dure, and maternal age >18 years. Multi-fetal pregnan-cies, ultrasonographically detected major fetal anomalies and chromosomal abnormalities, women with congenital uterine abnormalities, prior history of preterm birth or preeclampsia, thrombophilias, pre-pregnancy-diagnosed hypertension and diabetes, connective tissue diseases, any

smoking habit, women with BMI <18 or >28 and cases for which we could not attain delivery results were excluded. Pregnancy losses within 4 weeks of the amniocentesis, which were thought to be possibly procedure related, were also excluded. the gestational age of all pregnancies was assessed either by the last menstrual period or by an early ultrasound scan if there was ambiguity of more than a week.

counseling prior to amniocentesis was given to all fami-lies and written informed consent in all cases was obtained prior to the procedure. Following the procedure, all patients were assessed by a detailed ultrasound scan. no local anes-thesia or prophylactic antibiotherapy following the amnio-centesis procedure was implemented. After the procedure, in patients with rh-incompatibility, 300 µg anti-D IgG was implemented intramuscularly.

Some of the pregnant women did not continue their prenatal follow-ups and give birth at the same depart-ment. Data on pregnancy outcomes were obtained from the clinic-held records, and information of those who delivered outside our hospital was obtained through tel-ephone interviews. Patients were divided into two main groups: the control group that included patients who had no obstetrical complications (group 1); and the adverse obstetric outcomes (AOO) group that consisted of those who have at least one poor obstetric outcome (group 2). the poor obstetric outcomes were preterm birth (deliv-ery that occurred at <37 weeks of gestation), pregnancy-induced hypertension and preeclampsia, gestational dia-betes, FGr (below the 10th percentile), fetal macrosomia (neonates weighing more than 4,000 g) and stillbirth (a fetal death, antepartum or intrapartum, after 24 gesta-tional weeks).

Amniotic fluid assay of MMP-9 and Zn

After amniocentesis, a sample of amniotic fluid was transported to the genetic laboratory for fetal karyotype. the 4-cc amniotic fluid samples were stored until the assaying day at −80 °c in deep freeze in the biochemis-try laboratory. On the assaying day, the specimens were thawed at room temperature. Soluble materials were assayed within 4 h.

total MMP-9 levels in the AF samples were measured using the enzyme-linked immunosorbent assay (elISA) method by a Human MMP-9 Platinum elISA kit (eBi-oscience, Bender MedSystems, Wien, Austria). test measurements were performed using a Kayto rt 2100 c microplate reader (Kayto electronics, china). Zinc levels were measured by the atomic absorption method from the same materials by a Perkin-elmer A Analyst 800 atomic absorption spectrophotometer (Perkin-elmer Inc., USA).

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Statistical analysis

Statistical analyses were conducted using the Statistical Package for the Social Sciences for Windows 15.0 software (SPSS, chicago, Il, USA). Descriptive statistics were given as mean, standard deviation, median, min–max, frequency and percentage. the Kolmogorov–Smirnov test was used to evaluate whether the continuous variables were normally dis-tributed. For continuous variables the independent samples t test or Mann–Whitney U test were used as appropriate. Sta-tistical significance was determined by using the chi-square or Fisher’s exact test to compare nominal variables, inde-pendent samples t test for comparison between two groups and one-way analysis of variance (AnOVA) to compare data across multiple groups. A probability value <0.05 was con-sidered the minimum level of statistical significance. A two-sided p value was considered for all comparisons.

Results

A total of 117 singleton pregnancies, including 74 in the control group (group 1) and 43 in the adverse obstetric out-comes group (group 2), were included in this study.

Demographic properties of patients are shown in table 1. the mean of gestational age at birth (38.54 ± 0.968 vs. 36.40 ± 4.644, p = 0.005) and mean of birth weight (3,369.73 ± 377.51 vs. 2,987.62 ± 1,093.55, p = 0.033) were significantly lower in the group 2 than the group 1. However, the mean of maternal age at birth, newborn sex ratio, parity and cesarean delivery rates were similar between the groups. A comparison amongst groups, the mean, median, maximum, minimum and p values are shown for zinc results in table 2 and for MMP-9 results in table 3. MMP-9 and Zn results of patients in both the group 2 and AOO subgroups were compared with those of group 1. An analysis of tables 2 and 3 demonstrates that for Zn levels, none of the groups differ significantly from the group 1 while for MMP-9 levels both preterm birth group and group 2 were significantly different from the group 1 (p = 0.043 and p = 0.015, respectively). In six out of 13 patients involved in preterm birth group, preterm birth was due to PPrOM. MMP-9 and Zn results of these six pregnancies

were compared with both the group 1 (p = 0.149 and 0.239, respectively) and preterm birth group (p = 0.786 and 0.111, respectively) and no significant difference was observed.

Discussion

Matrix metalloproteinases constitute a large family of zinc-dependent enzymes that are directly implicated in

Table 1 Demographic features of patients

Values are given as mean and ± standard deviation

Bold values indicate statistically significant

Group 1 (n: 74) Group 2 (n: 43) p

Age at delivery (years) (mean ± SD) 33.07 ± 5.07 33.02 ± 5.38 0.966

Gestational weeks at birth (weeks) (mean ± SD) 38.54 ± 0.968 36.40 ± 4,644 0.005

Birthweight (g) (mean ± SD) 3,369.73 ± 377.51 2,987.62 ± 1,093.55 0.033

Parity 1.81 1.81 0.949

newborn male sex ratio (%) 55.9 62.7 0.057

cesarean birth ratio (%) 63.5 64.3 0.93

Table 2 Zinc levels of group 1 and group 2

Zn values are expressed as µg/dl

PTB preterm birth, GHT gestational hypertension, GDM gestational diabetes mellitus, FGR fetal growth restriction, FMac fetal macroso-mia, STB stillbirths

Mean Median Minimum Maximum p

Group 1 (n = 74) 49.68 39.50 8.00 186.00

PtB group (n = 13) 45.14 43.50 12.00 92.00 0.860

GHt group (n = 11) 49.75 34.00 21.00 148.00 0.516

GDM group (n = 11) 40.20 33.50 12.00 122.00 0.182

FGr group (n = 8) 44.37 42.50 17.00 83.00 0.679

FMac group (n = 10) 40.60 33.00 13.00 106.00 0.243

StB group (n = 4) 89.75 72.50 33.00 181.00 0.147

Group 2 (n = 43) 51.38 40.50 12.00 181.00 0.879

Table 3 MMP-9 levels of group 1 and group 2

MMP-9 values are expressed as ng/dl

Bold values indicate statistically significant

PTB preterm birth, GHT gestational hypertension, GDM gestational diabetes mellitus, FGR fetal growth restriction, FMac fetal macroso-mia, STB stillbirths

Mean Median Minimum Maximum p

Group 1 (n = 74) 15.18 12.28 2.32 65.14

PtB group (n = 13) 9.09 7.93 3.50 16.57 0.043

GHt group (n = 11) 12.76 14.88 5.99 17.62 0.812

GDM group (n = 11) 9.71 9.58 1.34 16.57 0.179

FGr group (n = 8) 8.31 7.24 2.50 16.85 0.052

FMac group (n = 10) 9.40 9.64 5.12 13.08 0.186

StB group (n = 4) 16.67 11.85 1.16 41.83 0.854

Group 2 (n = 43) 10.21 8.87 1.16 41.83 0.015

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multiple physiological and pathological processes. they are believed to participate in these functions primarily through degradation and remodeling of most components of the ecM. there is evidence that MMP-2 and MMP-9 expression within the decidua and other intrauterine tis-sues increases at term [5, 6]. An increase in MMP-9 lev-els within the fetal membranes seems to be triggered by labor and intraamniotic infection [16, 17]. However, all these studies were performed with the samples obtained in the third trimester from the pregnancies complicated by PPrOM or patients with active labor. A study conducted by Vadillo-Ortega et al. [7] included four patient groups, one of which was created with gestations in the early weeks, similar to our study. In this study, MMP-9 activi-ties were higher in amniotic fluid from normal-term preg-nancies with labor and those complicated by PrOM, than early pregnancies and term gestations without labor. this study suggests that gestations without labor and PrOM are associated with decreased levels of amniotic fluid MMP-9. However, in this study, perinatal outcomes of the patients whose amniotic fluid MMP-9 levels were meas-ured between 12th and 16th gestational weeks were not followed. Another study conducted by lavee et al. [15] assessed MMP-2 and tIMP-2 in second trimester amni-otic fluid of women with normotensive pregnancies and those who eventually develop preeclampsia. Higher levels were found in the amniotic fluid of women prior to pres-entation of preeclampsia. Kim et al. [18] investigated pre-term birth biomarkers in mid-trimester amniotic fluid and their study suggested that MMP-8 levels were significantly elevated in amniotic fluid in 16–19th weeks’ gestation. In our study, MMP-9 and Zn levels in amniotic fluid sampled between the 16th and 19th weeks of gestation were initially assessed retrospectively in terms of perinatal outcomes. We compared the amniotic fluid MMP-9 and Zn levels of the group 2 and its subgroups with the group 1. the MMP-9 levels of the group 2 and its preterm birth subgroup were significantly reduced when compared to the group 1. this result indicated that reduced amniotic fluid MMP-9 levels in the 16–19th gestational weeks might be helpful in the prediction of preterm birth and adverse obstetric outcomes. nevertheless, the reason why low-level MMP-9 correlates with preterm birth is not clear. the rise of MMP-9 in pre-term birth and PPrOM cases, suggested by previous stud-ies, occurs not at the beginning of the inflammatory process or in early gestational weeks, but emerges near the time of complication. Another important conclusion to be deduced from our low MMP-9 results is that the infectious or non-infectious inflammatory processes, which are thought to play an important role in the etiology of preterm birth or PPrOM, are acute events, not chronic processes begin-ning from the earlier gestational weeks. Hence, a variety of screening and treatment approaches for vaginal infections

in the early second trimester aiming at preterm birth proph-ylaxis do not seem likely to be a benefit.

Preterm birth still remains a leading cause of neonatal mortality and morbidity in developed countries [19]. Pre-dicting and preventing preterm birth is one of today’s most searched topics. For this purpose, an important part of the studies has focused on the MMP enzyme family. Most of the studies have been conducted with the materials sampled at the time of preterm or term labor from fetal membranes [4–6, 17, 20], decidua [5], placenta [4, 22] and amniotic fluid [7, 11, 21–23] and correlate to an MMP increase. Fol-lowing the beginning of contractions (term or preterm), the amount of most MMPs also increases in the feto-maternal interface [5]. After the contractions begin, the content of MMPs in the amniotic fluid increase in favor of MMP-9, and this is probably related to the loss of progesterone activity [24]. As a result, a short time before preterm or term labor, or in the time of labor, MMP-9 expression, in particular, increases in all feto-maternal tissues. In other words, the increase of MMP-9 actually shows an acute incident.

In the early stages of pregnancy, during the invasion and reconstruction of spiral arteries, degradation of ecM is essential and MMPs play a crucial role in this process [25]. A decrease has been reported in the expressions of MMP-2,3,7,9 proteins by trophoblasts both in preeclampsia and in FGr [11]. Although studies demonstrating the increase of MMP-9 levels in placenta and decidua of preeclamptic pregnancies exist [8, 26, 27], some other studies show a decrease [9, 10]. In our study, the period in which genetic amniocenteses were performed, a statistically significant difference was not detected between the amniotic fluid MMP-9 levels of the preeclampsia group and the group 1 (p = 0.812). even though in the FGr group, the amniotic fluid MMP-9 levels were lower compared to the group 1, the difference was not statistically significant (p = 0.052) and values have been found to be close to the statistical sig-nificance border. Significant results are likely to occur in case of similar studies made with larger patient groups.

Increased MMP activity has been detected in many dis-eases, including atherosclerosis, cardiovascular disease, and type 2 diabetes [28, 29]. In studies made with diabetic pregnancies [30] and diabetic pregnant animal models [13, 14], compared to controls, increased MMP levels in pla-centa during the implantation process and parturition have been documented. On the contrary, Stojanovic et al. [31] did not detect an increase in serum MMP-2 and MMP-9 levels in pregnant women with GDM. In our study, a signif-icant difference was not detected in amniotic fluid MMP-9 levels between the gestational diabetes group and group 1 (p = 0.179).

In fetal macrosomy, Johannsson et al. [32] detected increased MMP-9 production and activity in cord plasma

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in macrosomic neonates. In our study, a statistically signifi-cant difference was not detected between the amniotic fluid MMP-9 levels of the fetal macrosomy group and the group 1 (p = 0.186).

Stillbirths are one of the most poorly understood adverse obstetric outcomes and the etiopathogenesis still remains unclear. risk factors include advanced maternal age and some medical conditions, such as maternal diabetes, hyper-tension and obesity [33]. In the literature, a study research-ing the relationship between MMP-9 levels and stillbirths has not been found. In our stillbirth group, compared with the group 1, in terms of amniotic fluid MMP-9 and Zn lev-els, no statistically significantly difference was detected (p = 0.854).

Zinc in amniotic fluid increases during pregnancy and the most significant period of this rise is in the third tri-mester [34]. Opinions about the role of amniotic fluid zinc levels in high-risk pregnancies are conflicting and there are few studies on this subject. laitinen et al. [35] in fetal malformations, and Wood et al. [36] in 40 % of cases of open spina bifida showed the increase in amniotic fluid concentrations of zinc. Kynast et al. [37] demonstrated low levels of zinc in the amniotic fluid in FGr cases. the amniotic fluid zinc levels in preeclamptic women were significantly elevated in the 32nd gestational week, while it was significantly lower in the 40th week [37]. However, some studies could not determine any significant relation-ship between amniotic fluid zinc levels and poor obstetric outcomes [38]. We also could not detect a significant differ-ence in our study. When table 3 is analyzed, it is seen that although in stillbirth group both mean and median values are higher compared to the group 1, the difference is not statistically significant. the greatest reason for this is that stillbirth group consists of four pregnancies. to investigate whether any statistical difference exists, studies with larger series are needed. For stillbirths, the presence of elevated zinc levels in early gestational amniotic fluid seems likely to be an indication. Similar situations are shown in table 2.

We encountered very few prospective studies in litera-ture investigating the association between the second tri-mester amniotic fluid MMP-9 and zinc levels and obstetric outcomes [7, 15, 18].

Our study has some limitations. One limitation is that our study is an observational study designed in the cross-sectional style. Second, the sample size is relatively small and the low number of patients in some groups also affects the test results. For example, in the preterm partu-rition group, although amniotic fluid MMP-9 levels were found to be significantly lower compared with the group 1 (p = 0.043), the amniotic fluid MMP-9 levels of women who delivered preterm due to PPrOM were not statisti-cally significantly different compared to MMP-9 levels of both the preterm birth group (p = 0.786) and group 1

(p = 0.149). In subgroups with small patient number, inter-pretation power might be weakened by this low sample numbers. third, since patients in some groups had more than one adverse pregnancy outcome, they were included in another group at the same time, and this might have adversely affected the results.

Conclusion

As a result, we found that the amniotic fluid MMP-9 levels of patients who delivered preterm were significantly lower between the 16th and 19th gestational weeks. In addition, these results may indicate that suspected inflammatory events in the etiology of preterm births and PPrOM are not a consequence of a chronic process beginning in the early gestational weeks, but an acute event. even though our study is limited due to its low patient number, it might be adopted as a pioneer that may bring a different perspective and encourage investigators for further researches. Stud-ies with larger series are needed to determine the normal values of MMP-9 in amniotic fluid between these weeks of gestation and to detect the threshold value of amniotic fluid MMP-9 for the prediction of preterm parturition. In addition, the answer to the question why MMP-9 decreases in earlier gestational weeks in pregnant women who sub-sequently deliver early could be unveiled by these studies.

Conflict of interest none.

References

1. Fanjul-Fernandez M, Folgueras Ar, cabrera S, lopez-Otin c (2010) Matrix metalloproteinases: evolution, gene regulation and functional analysis in mouse models. Biochim Biophys Acta 1803:3–19

2. Yonemoto H, Young cB, ross Jt et al (2006) changes in matrix metalloproteinase (MMP)-2 and MMP-9 in the fetal amnion and chorion during gestation and at term and preterm labor. Placenta 27:669–677

3. Maymon e, romero r, Pacora P et al (2000) evidence of in vivo differential bioavailability of the active forms of matrix met-alloproteinases 9 and 2 in parturition, spontaneous rupture of membranes and intra-amniotic infection. Am J Obstet Gynecol 183:887–894

4. Xu P, Alfaidy n, challis Jr (2002) expression of matrix metal-loproteinase (MMP)-2 and MMP-9 in human placenta and fetal membranes in relation to preterm and term labor. J clin endo-crinol Metab 87:1353–1361

5. Goldman S, Weiss A, eyali V, Shalev e (2003) Differential activ-ity of the gelatinases (matrix metalloproteinases 2 and 9) in the fetal membranes and decidua, associated with labour. Mol Hum reprod 9:367–373

6. Mclaren J, taylor DJ, Bell Sc (2000) Increased concentration of pro-matrix metalloproteinase 9 in term fetal membranes overly-ing the cervix before labor: implications for membrane remod-eling and rupture. Am J Obstet Gynecol 182:409–416

Arch Gynecol Obstet

1 3

7. Vadillo-Ortega F, Hernandez A, Gonzales-Avila G et al (1996) Increased matrix metalloproteinase activity and reduced tissue inhibitor of metalloproteinases-1 levels in amniotic fluids from pregnancies complicated by premature rupture of membranes. Am J Obstet Gynecol 174:1371–1376

8. Matsushita S, narumiya H, Hirata M et al (1999) expression of matrix metalloproteinase (MMP), tissue inhibitor of metallopro-teinase (tIMP) and transforming growth factor-β1 (tGF-β1) in human placenta during preeclampsia. trophobl res 13:395–406

9. Kolben M, lopens A, Blaser J et al (1996) Proteases and their inhibitor are indicative in gestational disease. eur J Obstet Gynaecol reprod Biol 68:59–65

10. Shokry M, Omran OM, Hassan HI et al (2009) expressions of matrix metalloproteinases 2 and 9 in human trophoblasts of nor-mal and preeclamptic placentas: preliminary findings. exp Mol Pathol 87:219–225

11. reister F, Kingdom Jc, ruck P et al (2006) Altered protease expression by periarterial trophoblast cells in severe early-onset preeclampsia with IUGr. J Perinat Med 34:272–279

12. Merchant SJ, crocker IP, Baker Pn et al (2004) Matrix metal-loproteinase release from placental explants of pregnancies com-plicated by intrauterine growth restriction. J Soc Gynecol Investig 11:97–103

13. Pustovrh c, Jawerbaum A, Sinner D et al (2002) Metallopro-teinase 2 activity and modulation in the uterus from neonatal streptozotocin-induced diabetic rats during embryo implantation. reprod Fertil Dev 14:479–485

14. Pustovrh c, Jawerbaum A, capobianco e et al (2005) Increased matrix metalloproteinases 2 and 9 in placenta of diabetic rats at midgestation. Placenta 26:339–348

15. lavee M, Goldman S, Daniel-Spiegel e, Shalev e (2009) Matrix metalloproteinase-2 is elevated in midtrimester amniotic fluid prior to the development of preeclampsia. reprod Biol endo-crinol 7:85

16. Athayde n, edwin SS, romero r et al (1999) A role for matrix metalloproteinase-9 in spontaneous rupture of the fetal mem-branes. Am J Obstet Gynecol 179:1248–1253

17. Fortunato SJ, Menon r, lombardi SJ (1997) collagenolytic enzymes (gelatinases) and their inhibitors in human amniochori-onic membrane. Am J Obstet Gynecol 177:731–741

18. Kim A, lee eS, Shin Jc, Kim HY (2013) Identification of bio-markers for preterm delivery in mid-trimester amniotic fluid. Pla-centa 34(10):873–878

19. lawn Je, cousens S, Zupan J (2005) 4 million neonatal deaths: when? where? why? lancet 365:891–900

20. Hillier Sl, Martius J, Krohn M et al (1988) A case-control study of chorioamnionic infection and histologic chorioamnionitis in prematurity. n engl J Med 319:972

21. Maymon e, romero r, Pacora P et al (2000) Human neutrophil collagenase (matrix metalloproteinase 8) in parturition, prema-ture rupture of the membranes, and intrauterine infection. Am J Obstet Gynecol 183:94

22. Maymon e, romero r, Pacora P et al (2000) evidence for the participation of interstitial collagenase (matrix metalloprotein-ase 1) in preterm premature rupture of membranes. Am J Obstet Gynecol 183:914

23. Helmig Br, romero r, espinoza J et al (2002) neutrophil elastase and secretory leukocyte protease inhibitor in prelabor rupture of membranes, parturition and intra-amniotic infection. J Matern Fetal neonatal Med 12:237

24. Goldman S, Shalev e (2006) A proposed mechanism for proges-terone regulation of trophoblast MMP2 transcription independent of classical progesterone response elements on its promoter. J exp clin Assist reprod 3:4

25. Hulboy Dl, rudolph lA, Matrisian lM (1997) Matrix metal-loproteinases as mediators of reproductive function. Mol Hum reprod 3:27–45

26. Graham cH, Mccrae Kr (1996) Altered expression of gelatinase and surface-associated plasminogen activator by trophoblast cells isolated from placentas of preeclamptic patients. Am J Obstet Gynecol 175:555–562

27. Sankaralingam S, Arenas IA, lalu MM, Davidge St (2006) Preeclampsia: current understanding of the molecular basis of vascular dysfunction. expert rev Mol Med 8:1–20

28. Kai H, Ikeda H, Yasukawa H et al (1998) Peripheral blood levels of matrix metalloproteinase-2 and -9 are elevated in patients with acute coronary syndromes. J Am coll cardiol 32:368–372

29. Wang W, Schulze cJ, Suarez-Pinzon Wl et al (2002) Intracellular action of matrix metalloproteinase-2 accounts for acute myocar-dial ischemia and reperfusion injury. circulation 106:1543–1549

30. Pustovrh c, Jawerbaum A, Sinner D et al (2000) Membrane-type matrix metalloproteinase-9 activity in placental tissue from patients with pre-existing and gestational diabetes mellitus. reprod Fertil Dev 12:269–275

31. Stojanovic n, lewandowski K, Salata I et al (2010) Serum lev-els of matrix metalloproteinases (MMP)-2 and MMP-9 and their inhibitors in women with glucose intolerance in pregnancy and normal controls. Gynecol endocrinol 26:201–207

32. Johannsson e, Henriksen t, Iversen OI (2006) Increase in matrix metalloproteinases from endothelial cells exposed to umbili-cal cord plasma from high birth weight newborns. Am J Physiol regul Integr comp Physiol 292:1563–1568

33. Mcclure eM, Saleem S, Pasha O et al (2009) Stillbirth in devel-oping countries: a review of causes, risk factors and prevention strategies. J Matern Fetal neonatal Med 22:183–190

34. Brandes J, lightman A, ltskovitz J, Zinder O (1980) Zinc con-centration in gravida’s serum and amniotic fluid during normal pregnancy. Biol neonate 38:66–70

35. laitinen r, Jalanko H, Kolho Kl et al (1985) Zinc and alpha feto protein in amniotic fluid from early pregnancies with fetal mal-formations. Am J Obstet Gynecol 152:461–565

36. Wood BJ, Whyley GA, lawrence DM (1985) evaluation of amniotic fluid zinc levels in open neural tube defects. J Obstet Gynecol 5:215–218

37. Kynast G, Saling e, Wagner n (1979) the relevance of zinc determination in amniotic fluid: 2nd communication: zinc in cases of high fetal risk. J Perinat Med 7:69–71

38. Mahomed K, Grant D, James DK (1993) Amniotic fluid zinc and pregnancy outcome. eur J Obstet Gynecol reprod Biol 52:157–161