A novel optical method to assess cervical changes during pregnancy and use to evaluate the effects of progestins on term and preterm labor

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A novel optical method to assess cervical changes during pregnancy and use to evaluate the effects of progestins on term and preterm labor
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   A Novel Optical Method to Assess Cervical Changes duringPregnancy and Use to Evaluate the Effects of Progestins onTerm and Preterm Labor  Ruben J. Kuon 1,2 , Shao-Qing Shi, MD 1 , Holger Maul, MD 2 , Christof Sohn, MD 2 , JamesBalducci, MD 1 , Leili Shi, DDS 1 , and Robert E. Garfield, PhD 1 Department of Obstetrics and Gynecology of the St. Joseph’s Hospital and Medical Center,Phoenix, AZ, USAUniversity of Heidelberg, Germany  Abstract Objectives— To determine if optical methods can estimate cervix function during pregnancy and if progestins modify this process. Study Design— Photos of the external cervix of timed-pregnant rats were taken every other dayfrom day 13 (d13) until pp5 (postpartum day 5) following daily treatments with vehicle (controls)or progestin treatments (progesterone, P4 s.c., or vaginally; 17-alpha-hydroxyprogesteronecaproate, 17P s.c. and RU-486 s.c., once on d16). Surface area (SA) of the cervix was estimated from photos. Results— The SA of cervix increases throughout pregnancy and reverses pp in controls. In P4s.c. or 17P s.c. groups, increases in SA are lower (17P group until day 19 only) (P<.05). VaginalP4 does not prevent SA increases. Only the P4 s.c. blocked delivery. RU-486 increases SA of thecervix (P<.05) during preterm delivery. Conclusions— An optical method is useful for quantitative assessment of the cervix and evaluation of agents that modify cervical function. Keywords cervix; 17-alpha-hydroxyprogesterone caproate; preterm labor; progesterone; rats Introduction Preterm birth is a severe pregnancy complication that occurs in about 10 % of all pregnancies in the developed countries and even more often in developing countries.1, 2Despite all efforts, at this date reliable tools that predict and diagnose preterm birth as wellas successful treatment regiments for a better outcome of the pregnant woman and the babyare still missing. The development of effective therapies to prevent or reduce the occurrence © 2011 Mosby, Inc. All rights reserved.Reprints: Dr R.E. Garfield, PhD, Department of Obstetrics and Gynecology, St. Joseph’s Hospital and Medical Center, 445 N 5thStreet, Phoenix, AZ 85004. robert.garfield@chw.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Presented at the 30th Annual Meeting of the Society for Maternal-Fetal Medicine, Chicago, IL, Feb. 2–6, 2010.  NIH Public Access Author Manuscript  Am J Obstet Gynecol . Author manuscript; available in PMC 2012 July 1. Published in final edited form as: Am J Obstet Gynecol  . 2011 July ; 205(1): 82.e15–82.e20. doi:10.1016/j.ajog.2011.02.048. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    of this difficult medical condition depends on the understanding of the circumstances thatinitiate labor.After staying rigid and closed throughout most of pregnancy, to protect and secure thespecial environment created inside the uterus, the cervix switches to a soft and easy-to-openstate that is essential for successful vaginal delivery. Many biochemical and functionalchanges occur in the cervical connective tissue during gestation, which are summarized inthe term “cervical ripening”. It is a chronic process, which starts in the first trimester of  pregnancy and progressively proceeds until term. It is usually described as a 3-step preparation process, which must occur in sequence and each step seems to be irreversible:First softening, then effacement and finally dilatation of the cervix.3 This sequence isassociated with a dramatic reorganization of the extracellular matrix, which consists of elastin, proteoglycans and especially collagen that decreases by 30–70 % and is accountable by a change from insoluble to more soluble collagen.4, 5 Cervical ripening is an active biochemical process with similarities to an inflammatory like reaction (infiltration of leukocytes, increase of cytokines and metalloproteinases) and occurs independent of uterinecontractions.6 – 9 This process also appears to be at least partially regulated by steroid hormones (in particular progesterone (P4) and estrogen), as antiprogestins successfullyinduce cervical ripening.10 – 12 Other hormones and mediators shown to be involved incervical ripening are dihydrotestosterone13, prostaglandins14, and local mediators such as platelet-activating factor15 and nitric oxide14The consistent and precise identification of the changes that occur in the cervix is one of thechallenges obstetricians face today. Various methods have been used to identify thiscondition. Physical examination is one of the oldest techniques and a number of scoringsystems to characterize the cervix (e.g. the Bishop Score) have been developed.16, 17 Other techniques to assess cervical changes are the measurement of the cervical length bytransvaginal ultrasound and biochemical markers, like fetal fibronectin, a glycoprotein found at the chorionic-decidual interface, or Insulin-like growth factor binding protein-1, a proteinsynthesized by the maternal decidua.17 Our group has used light-induced fluorescence (LIF)of the cervix to estimate changes in cervical collagen and effects of treatments.18, 19 Thesestudies show that parenteral or topical P4 are equally effective in inhibiting delivery in rats but these treatments only partially prevent cervical ripening.19Recent studies have investigated the use of progestins as treatment for preterm delivery.20 – 26 Early studies also discussed the potential benefit of 17- α  hydroxyprogesterone caproate(17P), a synthetic caproate ester of the naturally occurring metabolite of P4, for thetreatment or prevention of preterm labor.27 Several recent randomized control trials havestudied the effects of progestins on cervical length changes assessed by transvaginalultrasound.24 – 26 Other possible treatments for woman at risk of cervical insufficencyinclude bed rest, cervical cerclage and antibiotics.28 – 30 None is evidence-based and there isa controversy in the findings. Uterine contractions can be suppressed by tocolytic drugs butonly for a very limited time and all compounds have considerable side-effects.31, 32Pregnant rats, a well-known model to study pregnancy in animals, are exquisitely sensitiveto changes in P4 with preterm delivery or prolonged gestation when P4 levels aremanipulated or P4 receptor antagonists are used.33 Our hypothesis is that exogenous progestins inhibit cervical ripening and prevent term delivery, that there are differences inabilities of the progestins and that consideration of routes of administration is important.The objective of this study was to determine if optical methods can be used to estimatechanges of the properties of cervical tissue during pregnancy and whether progestins given by various routes can alter these properties. We also used light-induced fluorescence to Kuon et al.Page 2  Am J Obstet Gynecol . Author manuscript; available in PMC 2012 July 1. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    assess cervical changes and compared them to an optical surface analysis of changes in areaof the cervix during pregnancy and postpartum. Study Design  Animals Timed-pregnant Sprague-Dawley rats (200–250 g) from Charles-River Laboratories(Wilmington, MA, USA) were transferred to our animal care facilities on day 12 of gestation (day 1 being the day when a sperm plug was observed). The animals were housed separately, with free access to food and water and maintained on a constant 12-hour light-dark cycle. Control pregnant rats were spontaneously delivering on day 22 and 23 of gestation. For the measurements with the endoscopic camera and the collascope the animalswere anaesthetized (i.p. injection) with a combination of xylazine (Gemini, BurnsVeterinary Supply Inc, Rockville Center, NY, USA) and ketamine HCl (Ketaset; Fort DodgeLaboratories Inc, Fort Dodge, IO, USA). The animals were randomly allocated to one of thegroups and sacrificed by carbon dioxide inhalation on day 5 postpartum or on day 25 of  pregnancy in the groups with delayed delivery. All procedures were approved by the AnimalCare and Use Committee of the St. Joseph’s Hospital and Medical Center in Phoenix. Treatments Pregnant rats (N = 6/ group) were treated, when not otherwise mentioned, from day 13 of  pregnancy until delivery. Single daily treatments were performed at 8 a.m. and twice a daytreatments at 8 a.m. and 8 p.m. All daily injections (4 mg P4 and 10 mg 17P) were by thesubcutaneous route (s.c.) in sesame oil (0.2 ml), which was also used for the controls of theinjection groups. Vaginal gels were applied twice a day with a blunt ball-top needle deepinto the vagina. Crinone was used for the P4 vaginal group (we used equivalent volumes of Crinone for 2–15 mg P4/ treatment, all data presented show the results of the highest dose(total daily dose of 30 mg P4 = 1/3 of a applicator of 8% Crinone that contains 90 mg P4).The control rats for the vaginal groups were treated with Replens (0.18 ml/ treatment).RU-486 (3 mg in 0.2 ml sesame oil) was injected s.c. once on day 16 of gestation. Reagents Crystalline P4 (used for subcutaneous P4), RU-486, sesame oil and ethanol were purchased from Sigma (St Louis, MO, USA). 17P was obtained from MP Biomedicals (Solon, OH,USA). P4, 17P, and RU-486 were dissolved in ethanol and then mixed with sesame oil.Crinone (micronized P4 in Replens, a bioadhesive gel, used for vaginal P4) and Replensfrom Columbia Laboratories (Livingston, NJ, USA).  Assessment of cervical changes Measurements with the endoscopic camera and the collascope were performed on everyother day starting at day 13 until day 21 of gestation and on postpartum day 3 and/or  postpartum day 5 and for some animals also on postpartum days 4, 8 and 10. Optical evaluation with an endoscopic camera— A small pediatric speculum wasinserted into the vagina of the anesthetized animal and always opened to a certain level(distance between the top and the bottom part of the end of the speculum was set to standard width of 9 mm, which was used for calibration of the measurements of the surface area). Anendoscopic camera was placed in front of the cervix at approximately 10 mm and photoswere taken of the cervix and ends of the speculum (Figure 1). The surface area (SA in mm 2 )of the cervix was calculated from digitized photographs by morphometric methods using the Kuon et al.Page 3  Am J Obstet Gynecol . Author manuscript; available in PMC 2012 July 1. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    software ImageJ 1.43, National Institute of Health, Bethesda, Maryland, USA (see athttp://rsbweb.nih.gov/ij/).34, 35 Evaluation with the collascope using light-induced-fluorescence— The amountof cervical collagen was evaluated by measurement of the autofluorescent properties of cross-linked collagen with a new prototype of an instrument, termed collascope (Reproductive Research Technologies, Houston, TX), as used previously with an earlier  prototype.15, 18 After insertion of a small speculum into the vagina of the anesthetized animal, the optical probe of the collascope was placed on the surface of the exocervix. The probe, which is connected to the main unit of the instrument by a fiber optic cable, deliversexcitation light (wavelength, 339 nm) onto the cervix and also carries the fluorescent light(mainly caused by pyridinoline cross-links of collagen with a maximum peak at 390 nm) back to the instrument to a charge-coupled device camera to display the full spectrum of fluorescence and analysis of the photons that are emitted by the cervix. The exposure timefor excitation was 100 msec. The average of 20 measurements of the detected fluorescentintensity (photon count) at 390 nm was used for each animal at any given time. Determining the changes in delivery time Pregnant animals were checked for delivery 3 times per day (8 a.m., 12 a.m., 8 p.m.). Theexpulsion of one pup was defined as the start of delivery.  Analysis The SA of the cervix and the LIF of control animals (Figure 2) obtained at different times of gestation were compared using one-way analysis of variance (ANOVA) and multiple pairwise comparison procedures (Dunn's Method for cervical SA and Holm-Sidak for cervical LIF). Student’s t-test was used to compare the SA of a treatment group to itsspecific control group at any time in gestation and postpartum (Figure 3) and also todetermine the differences in delivery times (Figure 4). A two-tailed probability value of P<0.05 was considered statistically significant. Results The SA of the cervix in normal pregnancy continuously increases throughout gestation (d13:12.6 ± 1.6 to 34.6 ± 3.7 at d21) and reverses pp (pp5: 12.2 ± 0.7) (Figure 2A).Measurements of cervical light-induced-fluorescence (LIF) in pregnant, non-treated animalsshow (Figure 2B) a continuously decreasing photon count throughout pregnancy and reversal postpartum. After significant (P<.05) changes from day 13 to day 15 the cervicalSA and the LIF reaches a wider plateau of non-significant (P>.05) changes prior to delivery.LIF values progressively increase postpartum (P<.05), whereas cervical SA decreasesrespectively (P<0.05). In parenterally treated P4 (Figure 3A) or 17P (Figure 3B) groupsincreases in SA are lower (P<.05) (d15/d19: P4-group: 15.3 ± 1.7/23.5 ± 3.4; 17P-group:16.0 ± 2.8 /24.2 ± 2.1; controls: 21.2± 3.9 /28.9± 4.2). Only for the P4-injection group is alower (P<.05) SA noted on d21 (P4 vs. control: 25.8 ± 4.6 vs. 34.6 ± 3.7). Vaginal P4(Figure 3C) does not prevent SA increases. RU-486 treatment increases the SA (P<.05)during preterm delivery (Figure 3D). Only parenteral P4 treatment blocked delivery (Figure4). Neither vaginal P4 nor parenteral 17P delayed delivery significantly (Figure 4). Comment This study demonstrates that an optical evaluation can be useful to assess quantitativechanges in cervical ripening in vivo. This method has never been described previously and may reveal a huge potential for the assessment of cervical changes in pregnancy as well as Kuon et al.Page 4  Am J Obstet Gynecol . Author manuscript; available in PMC 2012 July 1. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    for other gynecological conditions. The hereby introduced method is not only helpful toobserve the regular changes throughout pregnancy and postpartum, which have beeninvestigated in other studies using different techniques, but also indicates preterm cervicalchanges and the success of pharmacotherapy and interventions. As shown in this study progestins have the ability to delay cervical ripening and delivery in term pregnant rats.These effects depend critically on the choice of the progestin and the route of administration.Cervical ripening can be assessed by an optical examination of the exterior of the cervix.The SA of the external cervix increases almost 300% from day 13 (12.6 ± 1.6mm 2 ) of  pregnancy to term (day 21: 34.6 ± 3.7 mm 2 ) reflecting ripening (Figure 2A). Since wemeasure the overall surface area in photographs, not regarding folds, furrows etc. in theexterior of the cervix, it is undoubted that the absolute surface area is still underestimated and thus the method might be considered semiquantitative. Another method to assess earlychanges besides the optical evaluation is the use of light-induced fluorescence (LIF) of cross-linked collagen with an instrument called collascope.36 One of our previous studiesused LIF to assess cervical changes during pregnancy and the influence of progestins and the results and the conclusions support the present study.19 Throughout gestation thecollascope detects a decreasing photon count which describes the remodeling of theextracellular matrix including a decrease in collagen concentration and switchfrom_insoluble_to more soluble collagen (Figure 2B).18, 19, 37 This decrease of collagencould explain the softening of the cervix which is assessed by the endoscopic camera as anincrease in SA of the cervix in consequence. As anticipated in the postpartal period the LIFincreases progressively whereas the SA of the cervix decreases.As described previously the tremendous changes in the cervix occur early in pregnancy, inmidgestation. Following the concept of the 3-step process cervical ripening starts with a process called softening.3 This is a vital process and can not be assessed with the tools used at present in clinics to determine cervical problems in pregnancy. This optical evaluationreveals changes in the cervix earlier than many other techniques to assess the cervix.Ripening is associated with a strong reorganization of the extracellular matrix and anincrease of proteoglycans.7, 38 – 40 Related with this there is an influx of water into thetissue41 that may contribute to the increase in cervical SA.The increase in SA of the cervix is decelerated in the parenteral P4 (Figure 3A) and 17P(Figure 3B) groups and thus we conclude that these treatments delay cervical ripening butdo not entirely prevent it, indicating the involvement of other control pathways. In analogyto the comparison of the control groups the collascope again supports the results of theendoscopic camera in the same way for the treatment groups (results not shown).19 VaginalP4, even at 7.5 × the parenteral dose, does not inhibit ripening, as indicated in this study bychanges in the surface area of the cervix, or delivery possibly because of reduced P4 uptake(Figure 3C). Parenteral P4, but not 17P, inhibits delivery and may be more effective for treatment of preterm labor (Figure 4). As the cervix manages to ripen also in the parenteralP4 treated group at the end of gestation we conclude that the inhibition of delivery is not dueto an unripe cervix but must be due to an suppression of uterine contractions. Similar to the parenteral route we demonstrated in a previous study the block of delivery also for a topical(transdermal) route of administration of P4.19Softening of the cervix is a chronic process, whereas effacement and dilatation are acuteevents.3 Techniques that measure early pathological changes of the cervix before theirreversible steps of reorganization of the extracellular matrix are accomplished could be of great value in the identification of patients that are in high-risk for prematurity. This may bethe key why pharmacological interventions in clinical studies with P4 or 17P are notsuccessful or with contrary results: Women in danger of prematurity might be treated toolate when the drugs lose their influence to exert beneficial effects. Consequently many Kuon et al.Page 5  Am J Obstet Gynecol . Author manuscript; available in PMC 2012 July 1. 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