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J Pediatr Adolesc Gynecol (2009) -:-

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Congenital Imperforate Hymen with Hydrocolpos andHydronephrosis associated with Severe Hydramnios andIncrease of Maternal Ovarian Steroidogenic Enzymes

Emmanouil Karteris1, Helen Foster1, Maria Karamouti2, and Anastasia Goumenou1,2

1Centre for Cell Chromosome Biology, Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge, UK;2University of Crete, Division of Medicine, Department of Obstetrics and Gynecology, Heraklion, Crete, Greece

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Abstract. Study Objective: To study a clinical features ofpatient presented with severe hydramnios, associated withhydronephrosis, that was antenatally diagnosed and hasbeen successfully treated immediately after birth. At a mo-lecular level, we investigated the gene expression of keysteroidogenic enzymes from the maternal ovary.

Design: Ultrasound scan, MRI, semi-quantitative RT-PCRSetting: The patient was admitted to the University Hos-

pital, University of Crete, Medical School, Greece, whereall clinical data has been obtained. Gene expression studiestook place at Biosciences, Brunel University, UK.

Results: Semi-quantitative RT-PCR analyses revealedthat there is upregulation of key steroidogenic genes inthe maternal ovary, including steroidogenic acute regula-tory protein, and the cytochrome P450 heme-containingproteins CYP11A, CYP17 and CYP19. From a clinical per-spective, the prenatal ultrasound scan and MRI findingsshowed a multicystic pelvic mass, bilateral hydronephrosisand prior to delivery severe polyhydramnios.

Conclusion: This clinical case is the only one that wehave found in the current literature where congenital imper-forate hymen accompanied with hematocolpos is associatedwith renal obstruction in combination with polyhydramniosand increase in maternal steroidogenic enzymes.

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NCKey Words. Ovarian steroidogenic enzymes—Hyd

rocolpos—Polyhydramnios

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UIntroduction

Congenital imperforate hymen is the most frequentcongenital malformation of the female genital track,

Address correspondence to: Emmanouil Karteris, PhD, Centre for

Cell Chromosome Biology, Biosciences, School of Health Sci-ences and Social Care, Brunel University, Uxbridge, UB8 3PH,

UK; E-mail: Emmanouil.karteris@brunel.ac.uk

� 2009 North American Society for Pediatric and Adolescent GynecologyPublished by Elsevier Inc.

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TEDPRwhere mucus and blood from endometrial sloughing

accumulate in the vagina.1 When presented in adults,there is a distension of the vaginal canal that can leadto cervical dilation and formation of a hematometraand hematosalpinx. Symptoms in adults include cy-clic and abdominal pain, amenorrhea, and difficultywith urination.2 Retrograde menstruation, i.e., back-ward menstruation into the peritoneal cavity, can leadto the onset of endometriosis.

Endometriosis is a clinical and pathological entitythat is characterized by the presence of tissue that re-sembles functioning endometrial glands and stromaoutside the uterine cavity. Symptoms include infertil-ity and chronic pelvic pain.3 It has been hypothesizedthat vaginal secretions accumulate in response to cir-culating maternal estrogens.4 Interestingly, recentclinical and laboratory studies support the conceptthat endometriosis is an estrogen-dependent condi-tion. Indeed, high estradiol concentrations have beenidentified as a requisite for proliferation of endometri-otic lesions, and inducement of a hypoestrogenic stateis a current therapeutic approach.5

To the best of our knowledge, hydrocolpos (cysticdilatation of the vagina) as an isolated prenatal findinghas only been reported twice previously.6,7 Vaginalobstruction may be characterized by the sole presenceof an imperforate hymen, a transverse vaginal septum,or by the presence of a urogenital sinus or cloacalmalformation.8 Imperforate hymen is the result ofthe failure of canalization of the vaginal plate,1 whichis formed in part of the Mullerian ducts and from theurogenital sinus.8 A rare condition of congenital im-perforate hymen has been presented in which obstruc-tion of the vagina was associated with accumulationof uterovaginal secretions produced under circulatingmaternal estrogen stimulation, and the developmentof a hydrometrocolpos. Large hematocolpos may lead

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to secondary oligoamniosis, due to renal obstructionand deteriorating renal function. Interestingly, anadult case of hematocolpos was a consequence ofradiotherapy for cervix carcinoma.9 Moreover, in a re-cent study of a 15-year-old girl with imperforatehymen with hematocolpometra, the serum tumormarker CA125 was elevated.10

This particular clinical case is the only that we havefound in the current literature in which congenitalimperforate hymen accompanied with hydrocolpos isassociated with renal obstruction in combination withpolyhydramnios and increase in key steroidogeniccomponents of the maternal ovary.

Methods

Ovarian SamplesThe sample used in this clinical case was obtainedduring cesarean section surgery, due to the presenceof a cyst. Therefore, apart from the cyst, an ovarianbiopsy was also obtained from surface to clarify thenature of the cyst. The ovarian biopsy was taken withscissors and there was no significant bleeding. Allcontrol women were age matched, undergoing cesar-ean section, and there was a presence of ovarian cysts.For this study, maternal informed consent was ob-tained after the procedure was fully explained. Allprocedures followed were in accordance with the eth-ical standards of the responsible institutional commit-tee on human experimentation. Ovarian samples(n 5 4; 1 from the clinical case and 3 pooled controls)were placed in RNAlater� (Sigma Aldrich, UK) untilfurther use.

RNA Isolation and cDNA SynthesisEach sample was lysed in 600 ml of RNA lysis bufferusing the TissueLyser (Qiagen, UK). Total RNA wasextracted from these specimens using an RNA extrac-tion kit (Sigma, UK), according to manufacturer’s in-structions. RNA concentration was determined byspectrophotometric analysis (NanoDrop, Thermo Sci-entific, UK). RNA (200ng) was reverse-transcribed intocDNA using 5 IU/ml RNase H reverse transcriptase(Invitrogen, UK).

Semiquantitative RT-PCRPCR amplification was carried out using Taq poly-merase (Invitrogen). The primers used for thisstudy were: steroidogenic acute regulatory protein(StAR, 181 bp): 5’-CGTGACTTTGTGAGCG-3’ and5’-GCCACGTAAGTTTGGT-3’; CYP11A (202 bp):5’-AGAGTTGAAATCCAACA CC-3’ and 5’-TGGGACAGACGACTGA-3’; CYP17 (205 bp): 5’-GTGACCGTAA CCGTCT-3’ and 5’- ATGAACTGATCCGGCT-3’; HSD3B2 (298 bp): 5’-CCATACCCGTA-CAGCA-3’and 5’-AT TGACCTCGGACACT-3’;

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CYP19 (242 bp) 5’-CAGAGGCCAAGAGTTTGAGG-3’ and 5’-ACACTAGCAGGTGGGTTTGG-3’;b-actin (216 bp): 5’-AAGAGAGGCATCCTCACCCT-3’ and 5’-TACATGGCTGG GGTGTTGAA-3’.After an initial denaturation step of 94�C for 4 min,28 cycles were performed consisting of an initial de-naturing step at 94�C for 30s, followed by extensionat 60�C for 30 s and elongation at 72�C for 1 min.Densitometric analysis of resulted PCR products re-solved on a 2% agarose gel were quantified usingthe AlphaEase FC software.

Results

Molecular Findings—Changes in the GeneExpression of Steroidogenic Components in theMaternal OvaryAs mentioned previously, it has been hypothesizedthat vaginal secretions accumulate in response to cir-culating maternal estrogens.4 In this study we alsosought to investigate this hypothesis by assessingthe gene expression of key steroidogenic enzymesfrom the maternal ovary compared to age-matchedpooled controls. Semi-quantitative RT-PCR revealedsignificant upregulation of the following genes whencompared to an age-matched normal ovary: StARby 1.55, CYP11A by 1.67, CYP17 by 7.1 andCYP19 by 5.2 fold (Fig 1). The most profound in-crease was that of CYP17 and CYP19 when comparedto the normal ovary, whereas the levels of 3bHSD2 re-mained unaltered (data not shown). This is the firststudy that we have found to describe a fetus withhydrocolpos exhibiting changes in the expression ofmaternal steroidogenic enzymes at the ovarian level.

Clinical Findings—Identification of Cystic Massand Hydronephrosis Using Ultrasound Scan andSurgical ProceduresAn abdominal cystic mass with maximum diameter43.5� 29.4 mm was discovered on a prenatal sono-gram in a female fetus at 29 weeks gestation ina 32-year-old woman para 1, gravida 3. Fetal biome-try was within the normal range for gestational age.The patient’s prenatal course, which included amnio-paracentesis (because of patient’s family history fortrisomy 21 in a maternal aunt) had been uncompli-cated and previous ultrasound scan examinations aswell as 21 weeks ultrasound scan were reported nor-mal. Cytogenetic analysis revealed a normal femalekaryotype (46 XX), and no chromosomal abnormali-ties were detected. There was no history of familialimperforate hymen. All serial prenatal ultrasoundscans were performed at a specialist fetal medicineunit. On follow-up scans every 5e10 days, the ap-pearance of the pelvic cystic mass showed a gradualenlargement (Fig 2, panels a, b) and mild bilateral

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StAR

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Fig. 1. Changes in the gene expression of steroidogenic components in the maternal ovary. Lane 1: cDNA from normal ovaries;Lane 2: cDNA from patient; Lane 3: negative control.

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Fig. 2. Panel a: Longitudinal view of the large cystic ab-dominal mass with gravity dependent echoes, extendingup to the diaphragm at 33� 5 weeks gestation. Panel b:Transverse view at 33� 5 weeks gestation reveals a largecystic abdominal mass. Panel c: Sagittal view of bilateralhydronephrosis, at 33� 5 weeks gestation

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Fig. 3. A view of surgical hymenotomy performed by pedi-atric surgeons.

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Uhydronephrosis (Fig 2, panel c), but there were nochanges in amniotic fluid volume.

The patient was admitted to the hospital at 32weeks because of premature uterine contractions andcorticosteroids were administered to prepare for even-tual preterm birth as well as tocolytic therapy intrave-nously (IV) to control contractions. At 35 weeks ofgestation the size of cystic mass increased, measuring86� 75 mm maximum and hydronephorsis worsened.

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volume significantly increased (amniotic fluid index(AFI) was 25e27 cm) followed by hydramniosdevelopment. The estimated fetal weight was 3150 g.

Due to premature contractions and a history of pre-vious cesarean section, the patient underwent anemergency cesarean section and a female neonateweighting 3250 g, at 35 weeks of pregnancy, with Ap-gar scores of 8 and 9 after 1 and 5 minutes, respec-tively, was delivered. The neonate was admitted tothe neonatal intensive care unit for ventilatory supportand further investigations. Physical examinationshowed soft but distended mass above the umbilicusabdomen measuring 8� 7 cm.

Bladder catheterization was performed withdifficulty and clear urine was expressed. Renal func-tion tests were normal at birth, with a creatinine of0.7 mmol/L, and urea at 24 mmol/L. Abdominal ultra-sound examination showed a cystic mass posterior tothe bladder, bilateral hydronephrosis (1.3 cm) andhydroureter. MRI revealed an abdominal cystic masswith maximal diameter 90� 70 mm, which was feltto be a hydrometrocolpos due to an imperforatehymen.

Additional investigation of the urinary system dem-onstrated normal kidneys, renal calyces, ureter, anda small bladder displaced to the anterior abdominal walldue to the mass effect of the hydrocolpos. Neither refluxnor ureteroceles were observed. The patient was re-ferred to pediatric surgery. Consent was obtained fromthe baby’s parents, and a hymenectomy was performedwhere approximately 200 mL of clear serous andmucoid fluid was drained (Figs 3 and 4).

Discussion

Congenital imperforate hymen is the most frequentobstructive anomaly of the female genital tract. Thisgenerally occurs sporadically, with a reported

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Fig. 4. View of tubing for drainage of retained secretionstrough vagina.

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Eincidence at term11 of 0.014e1% and gives rise to hy-drometrocolpos in less than 1/16000 female births.12

Failure of this membrane to rupture results in congen-ital imperforate hymen, which in combination withaccumulation of uterovaginal secretions, (producedas a consequence of intrauterine stimulation of cervi-cal mucous glands by maternal estrogen hormones),may lead to the development of an internal hydrome-trocolpos.13 This can be presented as a pelvic mass incombination or not with urinary track obstruction andoligohydramnios.

In this clinical case the cystic mass presented for thefirst time during the 3rd trimester (29 weeks of gesta-tion), having hypoechoic content with a maximumdiameter of 43.5� 29.4 mm. At 35 weeks the cysticmass had a maximum diameter of 86� 75 mm causingbilateral urinary obstruction and hydronephrosis. What

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Fig. 5. Diagrammatic representation of steroid hormone biosynthethat have been upregulated in the maternal ovary of the fetus wi

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is interesting and unique in our case is the fact thatalthough there was a lack of a normal urinary outlet,oligohydramnios was not found. On the contrary, anevident increase of the amniotic fluid volume (AFI,25e27 cm) was observed. Cianciosi et al suggestedthe following mechanisms for accompanied hydram-nios in abdominal cystic masses: (a) the compressionof the adjacent bowel by the expanding cyst causinga hypoperistaltic intestine, or reduction of the absorp-tive capacity of the stomach and bowel by modifyingthe gastrointestinal vascularization, and (b) the depres-sion Qor an incorrect swallowing process, as suggestedby tongue protrusion, reducing the fluid removal fromthe amniotic cavity.14

The differential diagnosis of imperforate hymen isfrom labial adhesions, vaginal atresia, vaginal agene-sis, and transverse vaginal septum. However, the ab-dominal mass has to be diagnosed differentiallyfrom distental urinary bladder, ovarian tumors andneoplasms, mesenteric cysts, anterior meningoceles,reduplication of sigmoid and sacral tumors. Prenatalultrasonographic diagnosis of hydrocolpos has onlybeen reported in three cases,7,15,16 none of whichhad hydramnios detected.

As mentioned previously, it has been hypothesizedthat vaginal secretions accumulate in response tocirculating maternal estrogens.4 Therefore, based onour observations from the gene expression studies, itis attractive to speculate that, in this particular clinicalcase of hydrocolpos, the overall steroidogenic activityin the maternal ovary would have been enhanced. In-deed, when compared to control patients, the largestincrease in gene expression was that of CYP17 andCYP19. CYP17 is expressed in all classic steroido-genic organs and in the human ovary it is selectively

Q5sis steps in the human ovary. In red Q6are the key componentsth hydrocolpos.

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expressed in thecal cells.17 CYP19, on the other hand,has a far wider distribution and in the human ovary isprimarily expressed in the corpus luteum. This is ofparticular importance to this case because CYP19catalyzes reactions leading to estrogen biosynthesis.Depending on its expression, CYP19 (P450arom)catalyzes the conversion of the C19 androgens, an-drostenedione and testosterone, to the C18 estrogens,estrone and estradiol, respectively.17

It is well known that steroids such as maternalestrogens as well as progesterone rise exponentiallyduring the later stages in pregnancy. What is interestingin this clinical case is that the control ovarian sampleswere also taken from age-matched women, undergoingcesarean section. As a result, it is the possible dysregu-lation of the maternal steroidogenic pathway in thisclinical case that might contribute to the upregulationof certain enzymes. In addition, with regard to a link be-tween types of circulating estrogens and polyhydram-nios, very little is known. However, a study by Phocaset al showed that there was a discrepancy in the estrogenlevels regarding their increase. This was due to the typepolyhydramnios, the pregnancy outcome, and thecondition of fetus at birth.18

In conclusion, this is a novel clinical case where thereis clear evidence of a maternal steroidogenic over-driveassociated with fetal hydrocolpos, thus potentially link-ing maternal ovarian steroidal stimulation with thispathophysiological condition.

Uncited Figure

Fig 5

References

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2. Bakos O, Berglund L: Imperforate hymen and rupturedhematosalpinx: a case report with a review of the literature.J Adolesc Health 1999; 24:226

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3. Kobayashi H, Yamada Y, Kanayama S, et al: The role ofiron in the pathogenesis of endometriosis. GynecolEndocrinol 2009; 25:39

4. Yıldırım G, Gungorduk K, Aslan H, et al: Prenatal diagno-sis of imperforate hymen with hydrometrocolpos. ArchGynecol Obstet 2008; 278:483

5. Nothnick WB, Zhang X: Future targets in endometriosistreatment: targeting the endometriotic implant. Mini RevMed Chem 2009; 9:324

6. Adaletli I, Ozer H, Kurugoglu S, et al: Congenital imper-forate hymen with hydrocolpos diagnosed using prenatalMRI. AJR Am J Roentgenol 2007; 189:W23

7. Winderl LM, Silverman RK: Prenatal diagnosis of congenitalimperforate hymen. Obstet Gynecol 1995; 85:857

8. Rock JA, Breech LL: Surgery for anomalies of the Muller-ian ducts. In: Rock JA, Jones HW III, editors. Te Linde’sOperative Gynecology, (9th ed.). Philadelphia, LippincottWilliams & Wilkins, 2003, pp 705e749

9. Soloway MS, Rao MK, Kest L: Hematocolpos with urinarytract obstruction in an adult. J Urol 1977; 117:811

10. Kalmantis K, Koumpis C, Daskalakis G, et al: Imperforatehymen with hematocolpometra combined with elevatedCa125. Bratisl Lek Listy 2009; 110:120

11. Mor N, Merlob P, Reisner SH: Types of hymen in the new-born infant. Eur J Obstet Gynecol Reprod Biol 1986; 22:225

12. Benson CB, Doubilet PM: The fetal genitourinary system.In: Fleischer AC, editor. Sonography in Obstetrics and Gy-necology. London, Prentice-Hall International, 1996, pp 444

13. Spence HM: Congenital hydrocolpos. JAMA 1962; 180:1100

14. Cianciosi A, Mancini F, Busacchi P, et al: Increased amnioticfluid volume associated with cloacal and renal anomalies.J Ultrasound Med 2006; 25:1085

15. Davis GH, Wapner RJ, Kurtz AB, et al: Antenatal diagnosis ofhydrometrocolpos by ultrasound examination. J UltrasoundMed 1984; 3:371

16. Hill SJ, Hirsch JH: Sonographic detection of fetal hydro-metrocolpos. J Ultrasound Med 1985; 4:323

17. Payne AH, Hales DB: Overview of steroidogenic enzymesin the pathway from cholesterol to active steroid hormones.Endocr Rev 2004; 25:947

18. Phocas I, Salamalekis E, Sarandakou A, et al: Hormonal andbiochemical parameters in polyhydramnios. Eur J ObstetGynecol Reprod Biol 1987; 25:277

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