Congenital Imperforate Hymen with Hydrocolpos and Hydronephrosis associated with Severe Hydramnios and Increase of Maternal Ovarian Steroidogenic Enzymes

Semi-quantitative RT-PCR analyses revealed that there is upregulation of key steroidogenic genes in the maternal ovary, including steroidogenic acute regula- tory protein, and the cytochrome P450 heme-containing proteins CYP11A, CYP17 and CYP19. From a clinical per- spective, the prenatal ultrasound scan and MRI findings showed a multicystic pelvic mass, bilateral hydronephrosis and prior to delivery severe polyhydramnios.

This clinical case is the only one that we have found in the current literature where congenital imper- forate hymen accompanied with hematocolpos is associated with renal obstruction in combination with polyhydramnios and increase in maternal steroidogenic enzymes. Key Words. Ovarian steroidogenic enzymes—Hyd rocolpos—Polyhydramnios

Congenital imperforate hymen is the most frequent congenital malformation of the female genital track, where 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 lead to cervical dilation and formation of a hematometra and hematosalpinx. Symptoms in adults include cy- clic and abdominal pain, amenorrhea, and difficulty with urination. 2 Retrograde menstruation, i.e., back- ward menstruation into the peritoneal cavity, can lead to the onset of endometriosis. Endometriosis is a clinical and pathological entity that is characterized by the presence of tissue that re- sembles functioning endometrial glands and stroma outside the uterine cavity. Symptoms include infertil- ity and chronic pelvic pain. 3 It has been hypothesized that vaginal secretions accumulate in response to cir- culating maternal estrogens. 4 Interestingly, recent clinical and laboratory studies support the concept that endometriosis is an estrogen-dependent condi- tion. Indeed, high estradiol concentrations have been identified as a requisite for proliferation of endometri- otic lesions, and inducement of a hypoestrogenic state is a current therapeutic approach. 5 To the best of our knowledge, hydrocolpos (cystic dilatation of the vagina) as an isolated prenatal finding has only been reported twice previously. 6,7 V aginal obstruction may be characterized by the sole presence of an imperforate hymen, a transverse vaginal septum, or by the presence of a urogenital sinus or cloacal malformation. 8 Imperforate hymen is the result of the failure of canalization of the vaginal plate, 1 which is formed in part of the Mu ¨llerian ducts and from the urogenital sinus. 8 A rare condition of congenital im- perforate hymen has been presented in which obstruc- tion of the vagina was associated with accumulation of uterovaginal secretions produced under circulating maternal estrogen stimulation, and the development of a hydrometrocolpos. Large hematocolpos may lead 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: [email protected] /C2112009 North American Society for Pediatric and Adolescent Gynecology Published by Elsevier Inc. 1083-3188/09/$36.00 doi:10.1016/j.jpag.2009.10.002 J Pediatr Adolesc Gynecol (2009) -:- ARTICLE IN PRESS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 PEDADO1098_proof /C138 09–11–2009 23:14:21 UNCORRECTED PROOF to secondary oligoamniosis, due to renal obstruction and deteriorating renal function. Interestingly, an adult case of hematocolpos was a consequence of radiotherapy for cervix carcinoma. 9 Moreover, in a re- cent study of a 15-year-old girl with imperforate hymen with hematocolpometra, the serum tumor marker CA125 was elevated. 10 This particular clinical case is the only that we have found in the current literature in which congenital imperforate hymen accompanied with hydrocolpos is associated with renal obstruction in combination with polyhydramnios and increase in key steroidogenic components of the maternal ovary.

Ovarian Samples The sample used in this clinical case was obtained during cesarean section surgery, due to the presence of a cyst. Therefore, apart from the cyst, an ovarian biopsy was also obtained from surface to clarify the nature of the cyst. The ovarian biopsy was taken with scissors and there was no significant bleeding. All control 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. All procedures 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 /C210(Sigma Aldrich, UK) until further use. RNA Isolation and cDNA Synthesis Each sample was lysed in 600 ml of RNA lysis buffer using the TissueLyser (Qiagen, UK). Total RNA was extracted from these specimens using an RNA extrac- tion kit (Sigma, UK), according to manufacturer’s in- structions. RNA concentration was determined by spectrophotometric analysis (NanoDrop, Thermo Sci- entific, UK). RNA (200ng) was reverse-transcribed into cDNA using 5 IU/ ml RNase H reverse transcriptase (Invitrogen, UK). Semiquantitative RT-PCR PCR amplification was carried out using Taq poly- merase (Invitrogen). The primers used for this study were: steroidogenic acute regulatory protein (StAR, 181 bp): 5’-CGTGACTTTGTGAGCG-3’ and 5’-GCCACGTAAGTTTGGT-3’; CYP11A (202 bp): 5’-AGAGTTGAAA TCCAACA CC-3’ and 5’-TGGG ACAGACGACTGA-3’; CYP17 (205 bp): 5’-GTGA CCGTAA CCGTCT-3’ and 5’- A TGAACTGA TCC GGCT-3’; HSD3B2 (298 bp): 5’-CCA TACCCGTA- CAGCA-3’and 5’-A T TGACCTCGGACACT-3’; CYP19 (242 bp) 5’-CAGAGGCCAAGAGTTTGA GG-3’ and 5’-ACACTAGCAGGTGGGTTTGG-3’; b-actin (216 bp): 5’-AAGAGAGGCA TCCTCACC CT-3’ and 5’-TACA TGGCTGG GGTGTTGAA-3’. After an initial denaturation step of 94 /C14C for 4 min, 28 cycles were performed consisting of an initial de- naturing step at 94 /C14C for 30s, followed by extension at 60 /C14C for 30 s and elongation at 72 /C14C for 1 min. Densitometric analysis of resulted PCR products re- solved on a 2% agarose gel were quantified using the AlphaEase FC software.

Molecular Findings—Changes in the Gene Expression of Steroidogenic Components in the Maternal Ovary As mentioned previously, it has been hypothesized that vaginal secretions accumulate in response to cir- culating maternal estrogens. 4 In this study we also sought to investigate this hypothesis by assessing the gene expression of key steroidogenic enzymes from the maternal ovary compared to age-matched pooled controls. Semi-quantitative RT-PCR revealed significant upregulation of the following genes when compared to an age-matched normal ovary: StAR by 1.55, CYP11A by 1.67, CYP17 by 7.1 and CYP19 by 5.2 fold ( Fig 1 ). The most profound in- crease was that of CYP17 and CYP19 when compared to the normal ovary, whereas the levels of 3 bHSD2 re- mained unaltered (data not shown). This is the first study that we have found to describe a fetus with hydrocolpos exhibiting changes in the expression of maternal steroidogenic enzymes at the ovarian level. Clinical Findings—Identification of Cystic Mass and Hydronephrosis Using Ultrasound Scan and Surgical Procedures An abdominal cystic mass with maximum diameter 43.5 /C229.4 mm was discovered on a prenatal sono- gram in a female fetus at 29 weeks gestation in a 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 for trisomy 21 in a maternal aunt) had been uncompli- cated and previous ultrasound scan examinations as well as 21 weeks ultrasound scan were reported nor- mal. Cytogenetic analysis revealed a normal female karyotype (46 XX), and no chromosomal abnormali- ties were detected. There was no history of familial imperforate hymen. All serial prenatal ultrasound scans were performed at a specialist fetal medicine unit. On follow-up scans every 5 e10 days, the ap- pearance of the pelvic cystic mass showed a gradual enlargement ( Fig 2 , panels a, b) and mild bilateral 2 Karteris et al: Congenital Imperforate Hymen ARTICLE IN PRESS 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 PEDADO1098_proof /C138 09–11–2009 23:14:21 UNCORRECTED PROOF StAR CYP11A CYP17A CYP19A ββ-actin 1 2 3 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 Normal a e r cid l oFs 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 a e rc nidloF s 0 1 2 3 4 5 6 7 8 a erc n i d l oF s 0 1 2 3 4 5 6 a e r c n i d l oFs 0 1 2 3 e s a e r c nidloF Patient Normal Patient Normal Patient Normal Patient Normal Patient 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. Q13Karteris et al: Congenital Imperforate Hymen ARTICLE IN PRESS 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 PEDADO1098_proof /C138 09–11–2009 23:14:21 UNCORRECTED PROOF hydronephrosis ( Fig 2 , panel c), but there were no changes in amniotic fluid volume. The patient was admitted to the hospital at 32 weeks because of premature uterine contractions and corticosteroids were administered to prepare for even- tual preterm birth as well as tocolytic therapy intrave- nously (IV) to control contractions. At 35 weeks of gestation the size of cystic mass increased, measuring 86 /C275 mm maximum and hydronephorsis worsened. Ureteronephrosis was noticed while amniotic fluid volume significantly increased (amniotic fluid index (AFI) was 25 e27 cm) followed by hydramnios development. The estimated fetal weight was 3150 g. Due to premature contractions and a history of pre- vious cesarean section, the patient underwent an emergency cesarean section and a female neonate weighting 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 to the neonatal intensive care unit for ventilatory support and further investigations. Physical examination showed soft but distended mass above the umbilicus abdomen measuring 8 /C27 cm. Bladder catheterization was performed with difficulty and clear urine was expressed. Renal func- tion tests were normal at birth, with a creatinine of 0.7 mmol/L, and urea at 24 mmol/L. Abdominal ultra- sound examination showed a cystic mass posterior to the bladder, bilateral hydronephrosis (1.3 cm) and hydroureter. MRI revealed an abdominal cystic mass with maximal diameter 90 /C270 mm, which was felt to be a hydrometrocolpos due to an imperforate hymen. Additional investigation of the urinary system dem- onstrated normal kidneys, renal calyces, ureter, and a small bladder displaced to the anterior abdominal wall due to the mass effect of the hydrocolpos. Neither reflux nor ureteroceles were observed. The patient was re- ferred to pediatric surgery. Consent was obtained from the baby’s parents, and a hymenectomy was performed where approximately 200 mL of clear serous and mucoid fluid was drained ( Figs 3 and 4 ).

Congenital imperforate hymen is the most frequent obstructive anomaly of the female genital tract. This generally occurs sporadically, with a reported web 4C=FPO Fig. 2. Panel a: Longitudinal view of the large cystic ab- dominal mass with gravity dependent echoes, extending up to the diaphragm at 33 /C65 weeks gestation. Panel b: Transverse view at 33 /C65 weeks gestation reveals a large cystic abdominal mass. Panel c: Sagittal view of bilateral hydronephrosis, at 33 /C65 weeks gestation web 4C=FPO Fig. 3. A view of surgical hymenotomy performed by pedi- atric surgeons. 4 Karteris et al: Congenital Imperforate Hymen ARTICLE IN PRESS 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 PEDADO1098_proof /C138 09–11–2009 23:14:21 UNCORRECTED PROOF incidence at term 11 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 with accumulation of uterovaginal secretions, (produced as a consequence of intrauterine stimulation of cervi- cal mucous glands by maternal estrogen hormones), may lead to the development of an internal Q3 hydrome- trocolpos.13 This can be presented as a pelvic mass in combination or not with urinary track obstruction and oligohydramnios. In this clinical case the cystic mass presented for the first time during the 3rd trimester (29 weeks of gesta- tion), having hypoechoic content with a maximum diameter of 43.5 /C229.4 mm. At 35 weeks the cystic mass had a maximum diameter of 86 /C275 mm causing bilateral urinary obstruction and hydronephrosis. What is interesting and unique in our case is the fact that although there was a lack of a normal urinary outlet, oligohydramnios was not found. On the contrary, an evident increase of the amniotic fluid volume (AFI, 25e27 cm) was observed. Cianciosi et al suggested the following mechanisms for accompanied hydram- nios in abdominal cystic masses: (a) the compression of the adjacent bowel by the expanding cyst causing a hypoperistaltic intestine, or reduction of the absorp- tive capacity of the stomach and bowel by modifying the gastrointestinal vascularization, and (b) the depres- sion Q4or an incorrect swallowing process, as suggested by tongue protrusion, reducing the fluid removal from the amniotic cavity. 14 The differential diagnosis of imperforate hymen is from labial adhesions, vaginal atresia, vaginal agene- sis, and transverse vaginal septum. However, the ab- dominal mass has to be diagnosed differentially from distental urinary bladder, ovarian tumors and neoplasms, mesenteric cysts, anterior meningoceles, reduplication of sigmoid and sacral tumors. Prenatal ultrasonographic diagnosis of hydrocolpos has only been reported in three cases, 7,15,16 none of which had hydramnios detected. As mentioned previously, it has been hypothesized that vaginal secretions accumulate in response to circulating maternal estrogens. 4 Therefore, based on our observations from the gene expression studies, it is attractive to speculate that, in this particular clinical case of hydrocolpos, the overall steroidogenic activity in the maternal ovary would have been enhanced. In- deed, when compared to control patients, the largest increase in gene expression was that of CYP17 and CYP19. CYP17 is expressed in all classic steroido- genic organs and in the human ovary it is selectively web 4C=FPO Fig. 4. View of tubing for drainage of retained secretions trough vagina.web 4C=FPO Fig. 5. Diagrammatic Q5 representation of steroid hormone biosynthesis steps in the human ovary. In red Q6are the key components that have been upregulated in the maternal ovary of the fetus with hydrocolpos. 5Karteris et al: Congenital Imperforate Hymen ARTICLE IN PRESS 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 PEDADO1098_proof /C138 09–11–2009 23:14:22 UNCORRECTED PROOF expressed in thecal cells. 17 CYP19, on the other hand, has a far wider distribution and in the human ovary is primarily expressed in the corpus luteum. This is of particular importance to this case because CYP19 catalyzes 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 maternal estrogens as well as progesterone rise exponentially during the later stages in pregnancy. What is interesting in this clinical case is that the control ovarian samples were also taken from age-matched women, undergoing cesarean section. As a result, it is the possible dysregu- lation of the maternal steroidogenic pathway in this clinical case that might contribute to the upregulation of 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 Phocas et al showed that there was a discrepancy in the estrogen levels regarding their increase. This was due to the type polyhydramnios, the pregnancy outcome, and the condition of fetus at birth. 18 In conclusion, this is a novel clinical case where there is clear evidence of a maternal steroidogenic over-drive associated with fetal hydrocolpos, thus potentially link- ing maternal ovarian steroidal stimulation with this pathophysiological condition. Uncited Figure Fig 5

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