Pelvic Imaging in Reproductive Endocrinology

CHAPTER 35

Pelvic Imaging in Reproductive Endocrinology Dominique de Ziegler Isabelle Streuli Pietro Santulli Charles Chapron

Vaginal Ultrasound and In Vitro Fertilization The advent of vaginal ultrasound a quarter century ago revolutionized imaging of the pelvic cavity and in particular visualization of the ovaries and uterus. A limiting prinicpal in ultrasound is that lower frequency ultrasound has greater penetration (less attenuation) but lower spatial resolution, while higher frequency ultrasound has lower penetration (greater attenuation) but greater spatial resolution. Vaginal ultrasound takes advantage of the ability to place a probe close to the structures of interest. This permits the use of higher frequency ultrasound and improves the resolution over that possible with lower frequency abdominal ultrasound probes. The original purpose of vaginal ultrasound was not diagnostic imaging, but to simplify oocyte aspiration for in vitro fertilization (IVF), which was originally performed by a transvesical route.1 To facilitate this process, probes were designed for vaginal use and equipped with needle holders to permit transvaginal oocyte aspiration.2-4 The practical advantages of vaginal ultrasound for guiding oocyte retrieval were obvious and it rapidly became the primary mode of oocyte retrieval.5 Improvements in image resolution that resulted from the use of high-frequency probes sparked an instant interest for use of vaginal ultrasound in general gynecology.6-8 Subsequently, all the technological improvements incorporated into general ultrasound equipment, such as pulsed, color and power Doppler functions,9,10 and later, three-dimensional (3D) capabilities11,12 became available on vaginal probes. Ultimately, the increase in image resolution provided by vaginal ultrasound and its multiple refinements allowed for meticulous assessment of parameters that reflect the anatomical characteristics of pelvic organs, but also their functional status.13 For example, the antral follicular count (AFC) stands at par with hormonal ­parameters (anti-müllerian hormone [AMH] and/or day-3 follicle stimulating hormone [FSH] and estradiol [E2] levels) for assessing the functional state of the ovaries or ovarian reserve, and vaginal ultrasound is the main tool for monitoring ovarian response and follicular growth in controlled ovarian stimulation (COS).14

Vaginal Ultrasound and Pelvic Examination Beyond improving image resolution and quality, it rapidly became evident that vaginal ultrasound could provide information about functional interactions that exist between pelvic organs and nearby structures. This possibility stems from the fact that a vaginal ultrasound examination, as opposed to abdominal ultrasound, does not require a full bladder preparation. Consequently, it is possible to freely slide pelvic organs against one another, and move them about the pelvic cavity under direct ultrasound vision by gentle pressure applied to the vaginal probe. This approach offers a glimpse of the functional status of the pelvic cavity not available with static images. • It can precisely determine the limits of pathological structures, such as an ovarian cyst. This is done by determining how the identified structure—the ovarian cyst in our example—moves with respect to other nearby organs when pelvic structures are mobilized by the vaginal probe. At times, the help of pressure exerted by an “abdominal hand” may be necessary in order to mobilize the organ(s) being studied. • The ability to slide organs also provides clues about the possible presence of pelvic adhesions. For example, the ovary and nearby loops of bowel may move together when mobilized by the probe. • While pelvic organs are mobilized by pressure exerted either by the vaginal probe and/or with the help of an abdominal hand, the examiner can assess pain that this maneuver may generate. The dynamic vision provided by vaginal ultrasound allows the examiner to pinpoint the cause of the pain and its anatomical location. This functional dimension provides a true pelvic examination with a view, fusing various forms of information to provide a global assessment of pelvic status.

Technical Refinements of Vaginal Ultrasound: Doppler and 3D Reconstruction As vaginal ultrasound progressively became a primary diagnostic tool in gynecology, two technical refinements have 851

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come to enrich its capabilities and scope: Doppler-based analyses of organ perfusion and the off-line and later built-in 3D image reconstruction systems. In the case of 3D image reconstruction from saved 3D volumes, the technical novelty also offers new possibilities for improving the quality control of diagnostic ultrasound through secondary exploration of saved 3D volumes.

Contrast-Enhanced Vision of the Uterus and Tubes It struck many that the ultrasound images of the uterine cavity obtained in early pregnancy largely surpass in quality those of the nonpregnant uterus. The improved image quality of the pregnant as opposed to nonpregnant uterus was attributed to the presence of amniotic fluid, playing the role of a contrast enhancer that facilitates embryo visualization. Being sono-transparent like water, amniotic fluid creates a black interphase between the walls of the gestational sac and the developing embryo, which enhances image resolution. To reproduce the early pregnancy conditions, infusion of isotonic saline or other solutions into the uterine cavity of the nonpregnant uterus was introduced to create greater contrast. This procedure has several designations including saline infusion sonography (SIS), hysterosonography (HySo), or hysterosalpingocontrastsonography (HyCoSy).15,16 Numerous variants of the original procedure have been proposed that employ either sono-transparent17,18 or sono-refractory contrast media, such as Echovist®, Levovist® or Albumex®,19-21 which provide black and white contrast enhancement, respectively. Black contrast exploration of the uterine cavity has been found to be particularly helpful for exploring uterine malformations,22 favorably comparing with magnetic resonance imaging (MRI).23 While black contrast is superior for exploring the uterine cavity, white contrast offers the advantage of imaging the fallopian tubes.21 Another approach to contrast-enhanced ultrasound utilizes microbubbles manufactured with a hydrophilic shell of albumin, galactose, lipids or polymers and a hydrophobic gas core. When the gas bubbles interact with ultrasound energy, they alternately compress and expand, generating a strong, unique signal. Ultrasound devices can be specially tuned to enhance the detection of these unique signals.

Endometriosis and Adenomyosis: Ultrasound and Magnetic Resonance Imaging (MRI) Endometriosis can affect all pelvic organs, associated with various degrees of extension and infiltration. Exploring endometriosis often requires MRI. Likewise, a variant of endometriosis, adenomyosis—the diffuse and focal forms— can be reliably diagnosed on MRI with good correlation with anatomical findings.

Functional Pelvic Imaging in Reproductive Endocrinology and Infertility Anatomy and Morphological Measurements The two primary constituents of the uterus—the outer muscle or myometrium and its mucosa, the endometrium—present an

interphase that is easily identifiable on ultrasound. The lower end of the endometrium delineates the internal os of the cervix, which provides a landmark for individual measurements of the cervix and uterine corpus. The total length of the cervical canal is measured after identifying the external os of the cervix. The endometrium, which is hormonally sensitive, changes in thickness, volume, and aspect (echogenicity) during the menstrual cycle, and in response to hormonal treatments. The myometrium is composed of three layers.24,25 The inner, or sub endometrial layer of the myometrium, is of müellerian origin. It is endowed with estrogen receptors (ER) and progesterone receptors (PR) showing cyclical changes that mimic those encountered in the endometrium.26 The two outer nonmüllerian layers of the myometrium have constant concentrations of ER and PR throughout the menstrual cycle.24 In the nonpregnant uterus, the outer layer of the myometrium participates in uterine contractions encountered at the time of menses in response to progesterone withdrawal, but not in the retrograde contractions seen in the late follicular phase of the menstrual cycle. Disturbances of the contractile processes at the time of menses may cause dysmenorrhea or endometriosis, whereas alterations in the follicular phase may interfere with sperm transport.27 Current imaging is capable of identifying the subendometrial layer of the myometrium where the peristaltic contractions that culminate in the late follicular phase are initiated. High-resolution vaginal ultrasound defines this myometrial segment, the intermediate layer of lighter echogenic appearance, or subendometrial halo. It lies between the echoic basal layer of the endometrium and the underlying myometrium. Similarly, but using a different terminology, MRI identifies the subendometrial layer of the myometrium as a hypointense layer that separates the endometrium from the myometrium on T2-weighed images or the junctional zone (JZ) (see section on endometriosis and adenomyosis).28 Video recordings have identified that uterine contractions during the late follicular phase under the stimulus of rising E2 levels originate from the subendometrial halo or JZ area.29,30 In an ex-vivo trial, Lesny and colleagues29 studied six uteri obtained from fertile women 32.9 to 48.5 years old. Following hysterectomy, all uteri were needle-biopsied in the subendometrial halo under ultrasound guidance. The results indicate that the subendometrial halo, or JZ area, is a distinct section of the myometrium with increased ­vascularitiy and more tightly packed myometrial cells that is now also identifiable on ultrasound.31 As discussed later, endometriosis and adenomyosis are thought to be associated with dysfunction of the endometrial-subendometrial unit.24 The boundaries between the cervix and the uterine corpus, and between the endometrium and myometrium, are easily identifiable, so that the uterus can be measured on ultrasound scans (Fig. 35.1). In adult women, uterine length ranges from 5 to 8 cm32 following growth at the time of puberty. In a prospective trial examining 139 girls between the ages of 1 and 13 years of age with radiological bone age measurements, uterine volume was calculated from transabdominal scan images taking the measured values of length, width, and depth and using the ellipse formula.33 Uterine measurements and volume were markedly smaller in pretelarchial girls at 1.8 ± 1.2 cm3 as compared to 8.1 ± 6.6 cm3 after telarche.34 In a large prospective trial conducted on 380 school girls 6 to18 years of age, Holm

CHAPTER 35  Pelvic Imaging in Reproductive Endocrinology

FIGURE 35.1  Uterine size. The interphase between the endometrium and myometrium is identifiable on ultrasound images. The lower end of endometrial development identifies the internal os of the cervix, which delineates the limits of the uterine corpus and cervix. The yellow marks define the cavity interphase in the inner cervix and uterine corpus. The two outer marks define the outer uterine limits at the level of the corpus.

and colleagues observed no age-related differences in uterine and ovarian size in 44 prepubertal girls, whereas a direct correlation with Tanner stages was observed in 163 prepubertal girls.35 A further enlargement of the uterus was observed between Tanner stage 3 and adult girls (mean age of 19). Looking at 114 premenarchial girls, Orsini and colleagues observed that until the age of 7, the uterus and cervix were of similar size with the corpus gradually becoming larger than the cervix thereafter.36 These authors observed that the uterus continued to grow after menarche for several years, with uterine size correlating with the number of postmenarchial years, but not with height, a finding that has been challenged by others.35 There are clinical conditions in which the uterus was believed to be smaller than normal, a finding not always verified by measurements. Doerr and colleagues report on a series of 75 women affected by Turner syndrome. Of 50 women with a karyotype 45,X0 who had received estrogen replacement, 42 (84%) had a normal size uterus, whereas it was smaller (length less than 5 cm) in the remaining 8 women (16%).37 In this study, uterine size might have been affected by the age at which estrogen therapy was initiated, but showed no correlation with other factors, notably the final height.37 The impact of growth hormone (GH) treatment on uterine size remains a matter for debate, with Sampaolo and colleagues38 showing a positive effect whereas, Snajderova and colleagues found none.39 We know from Herbst and colleagues40,41 that in utero exposure to diethylstilbestrol (DES) may affect the genitalia. These authors linked seven cases of adenocarcinoma of the vagina, an otherwise excessively rare tumor, with in utero DES exposure. All these cases occurred at a single hospital in DES-exposed women between the ages of 7 and 22 years,41 a finding later confirmed by others.42 Further studies of DES-exposed women revealed many benign morphological abnormalities of the genital tract. These abnormalities, stemming from epigenic alterations43 of the HOX

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FIGURE 35.2  Effects of in utero exposure to diethylstilbestrol (DES): T-shaped uterus with characteristic mid-uterine ridges.

gene family44 include transverse ridges in the vagina, vaginal adenosis, and morphological anomalies of the uterus, such as the characteristic T-shaped uterus (Fig. 35.2).45 Viscomi and colleagues analyzed the echographic appearance of the uterus in cases of DES exposure.46 An abnormal T-shape uterus was found in 3 of the 18 (16%) women exposed to DES, a percentage in agreement with an incidence of 12% observed on hysterosalpingogram (HSG).47 Furthermore, Viscomi and colleagues reported a reduced uterine size with a mean uterine length of 6.8 ± 0.4 cm in 18 DES-exposed women as compared to 8.1 ± 0.8 cm in 20 age-matched controls, even in the absence of gross morphological anomalies.47 Calculated uterine volume was 49.4 and 90 cm3 in DESexposed women and controls, respectively. Salle and colleagues have reported altered uterine vascularity in women exposed to DES, with higher uterine artery pulsatility (PI) and resistance index (RI), and an absence of the cycle related changes.48 However, the true impact of these uterine flow differences are difficult to interpret. Several authors have proposed surgical correction (metroplasty) of uterine anomalies related to DES exposure, including T-shaped distortion49 or simple overall reduction of uterine size.50 Although authors reporting on small series recount positive impressions about the impact of surgery on subsequent reproductive outcome,51 the efficacy of these procedures remains to be rigorously evaluated.

The Uterine Cervix Vaginal ultrasound studies can precisely delineate the anatomical limits of the cervix. The cervical canal can easily be identified. When aqueous mucus is produced—under the influence of E2 in the follicular phase—the cervical canal appears sono-transparent and hypoechoic. Early in the luteal phase, the mucus changes its ionic constitution under the influence of progesterone and the canal appears hyperechogenic (white) on ultrasounds. We use a semiquantitative grading system in which hyperechogenic mucus is either not seen (-) or seen and graded as +, ++, and +++ when a sonotransparent dilatation of the cervical canal can be measured at less than 1 mm, 1 to 2 mm,

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and greater than 2 mm, respectively, an approach close, if not similar, to that of others.52 The presence of cervical mucus visible on ultrasound serves as a biomarker for estrogen exposure, as well as an indicator of the absence of progesterone effects.21 This latter parameter is particularly helpful when following patients treated with assisted reproductive technologies (ART). Identifying sonotransprent (black) cervical mucus on ultrasound makes it possible to disregard increases in plasma progesterone levels. This biomarker of progesterone’s effects is particularly handy when monitoring ovarian responses.21 An abrupt disappearance of cervical mucus observed concomitantly with plasma progesterone elevation in late stages of controlled ovarian hyperstimulation (COH), as sometimes encountered in poor ovarian responders, is an ominous sign.53 The sudden disappearance of cervical mucus on ultrasound associated with pre-human chorionic gnonadotropin (hCG) progesterone elevation constitutes grounds for canceling an ART cycle, or freezing embryos and deferring transfer.54 Changes in echographic characteristics of the cervix have received little attention.55 As expected from visual examination of the outer cervix, 3D assessment of the whole cervix reveals size differences between nulliparus and multiparous women.56 Links between cervical length and the risk of preterm labor (PTL) and delivery have been evaluated with new image-based screening for PTL.57-63 More pertinent to infertility is the early identification of cervical insufficiency and its corollary, clinical indications for preemptive cervical cerclage. This clinical topic remains controversial, in spite of the quality cervical length measurements and the number of well designed studies. A well-established indication for cervical length measurement in the decision for cervical cerclage is during the midtrimester in women with a past history of pregnancy losses due to cervical insufficiency and prior short cervix measurements on ultrasound.64 Various technical refinements may help in assessing cervical length using sonoelastography65 or harmonic imaging.66 A recent study indicated that assessing cervical length for reinforcing an existing cerclage may actually be counterproductive.67

The Endometrium: Biomarker of the Hormonal Environment Endometrial Thickness: a Biomarker for Estrogen Action By convention, the endometrial thickness is measured on ultrasound from one myometrial-endometrial interphase to the next, on the thickest point of the endometrium (Fig. 35.3). This widely accepted practice amounts to measuring a double endometrial thickness, something that has become a universally acepted convention. From being relatively thin at the time of menses, the endometrium progressively thickens during the proliferative phase of the menstrual cycle, commonly peaking at 7 to 9 mm on the day of the luteinizing hormone (LH) surge.68-70 Prior to ovulation, the endometrium takes on a multilayered or three line appearance formed by the echogenic basal layers and the two hypoechoic functional layers, separated by the hyperechoic interphase of the virtual uterine cavity (Fig. 35.4). The increase in endometrial thickness seen

FIGURE 35.3  Endometrial thickness. Measurements are made from one endometrial to myometrial interphase to the one on the opposite side, therefore amounting to a double endometrial thickness measurement.

FIGURE 35.4  Hypoechogenic or type I endometrium: Echographic appearance of the endometrium during the follicular phase. Between the hyperechogenic layer of the endometrial basalis and the echogenic line of the virtual endometrial cavity lies a characteristically hypogenic endometrium. Taken together, this constitutes the characteristic three-layer pattern of the proliferative endometrium. On this figure one can also identify mucus present in the cervical canal. With our semi-quantitative grading system, this corresponds to a +++ score for the quantity of mucus because the mean diameter of the dilated cervical canal is dilated at 2.2 mm.

throughout the follicular phase represents the endometrial proliferation induced by E2. This effect of estrogen, leading to the deployment of ER and PR, primes the endometrium for its response to progesterone. Under the influence of E2, endometrial glands develop vertically with strait lumens. The donor-egg ART model revealed the respective roles of E2 and progesterone in the priming of endometrial receptivity. Donor-egg ART taught us, in particular, the exceptional leeway that exists for the duration of the E2 priming phase, which can range from 1071 to 100 days without consequences on ART outcome.72 Provided that E2 priming is sufficient,54 the duration of E2 priming duration has

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little impact on endometrial thickness or other endometrial parameters of estrogen effects on the uterus such as contractility.73 As discussed later in this chapter, an endometrial thickness of ≥7 mm, or a volume of 2.9 mL,74,75 is seen as reflecting sufficient endometrial priming by E2. Extending treatment duration rather than increasing E2 administration is seen as the better option for managing the excessively thin endometrium.76 McWilliams and Frattarelli looked at the dynamic changes in endometrial thickness in ART rather than static data (endometrial thickness at a single given point).77 Fresh IVF cycles were studied in which the changes in endometrial thickness were analyzed from baseline to day 6 and from there to the day of hCG administration. Increments in endometrial thickness from baseline to day 6 and from day 6 to day of hCG were of 3.6 ± 2.4 and 2.0 ± 2.2 and of 2.3 ± 2.6 and 2.5 ± 2.1 in women who became pregnant (n = 70) and those who did not (n = 62), respectively. This comparison suggested that increments in endometrial thickness from baseline to day 6 were more predictive of pregnancy since there were no differences in the increment taking place from day 6 to the day of hCG. These dynamic changes in endometrial thickness point out the positive bias that exists with the quality of the ovarian response to COS, which can be wrongly interpretated as reflecting endometrial receptivity. This, therefore, constitutes a bias that must be remembered when interpreting an apparent link between endometrial thickness and endometrial receptivity78 drawn from analyses of ART data. Ng and colleagues studied standardized endometrial preparations used for frozen embryo transfers (FET).78a These authors found no differences in endometrial thickness or Doppler-assessed blood flow between pregnant and nonpregnant women after receiving similar E2 ­priming,78a a finding ­confirmed by others.79 Comparisons of endometrial thickness in the late follicular phase of the menstrual cycle, following physiological E2 and progesterone replacement or mild ovarian stimulation for FET revealed similar findings.80,81 This suggests that similar forms of hormonal priming have similar effects on endometrial thickness. Furthermore, exposure of the endometrium to E2 levels that are more than 10 times higher than menstrual cycle values, as encountered in ART82 and following the vaginal administration of 2 mg of E2/day83 barely resulted in a 20% thicker endometrium. This suggests that E2 priming that results from menstrual cycle levels of E2 is nearly maximum. As noted earlier, the same leeway that exists for the amount of E2 used for endometrial priming is also seen for the duration during which the priming is applied. In a retrospective analysis of their donor egg data, Pellicer’s group showed that extending the E2 priming phase for up to 100 days had no significant impact on ART outcome84 and endometrial thickness72 a finding confirmed by others.85,86 The minimal effects of increased and/or prolonged E2 priming on endometrial thickness conflicts with reports indicating that excessive ovarian responses to COS negatively impacts on pregnancy and embryo implantation rates.84,87 To account for these divergent findings, we proposed that in strong responses to COS, it is the excessive production of ovarian factors other than E2 that are responsible for any adverse endometrial effect, not a direct action of high E2 levels per se.54,88

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A consensus exists today that women whose endometrium is less than 7 mm in ART have markedly decreased chances of becoming pregnant,89,90 particularly if no triple line pattern was seen.91 In rare cases, albeit disturbing ones, this condition may persist, and be found in menstrual cycles and stimulated and E2-supplemented cycles.92 This condition is typically resistant to markedly elevated estrogenic exposure.83 It may be encountered in the aftermath of total body irradiation or other cancer treatments,93 or in the absence of readily available explanations. The treatments that have been proposed for these women include low dose aspirin,94 an estriol challenge test,95 locally active vasodilators,96 and the combination of pentoxifylline and tocopherol (vitamin E).92,97 The latter products, pentoxifylline and tocopherol, were tested on the grounds that they had been reported to be effective at reducing fibrosis induced by radiation therapy.98 While some reports speak for possible improvements in results in donor-egg ART recipients,99 no rigorous evidence for efficacy exists. The possible poor predictive value of a thickened endometrium in ART reported by Casper’s team100 remains a matter for debate. While confirmed by some,89 a larger group of publications failed to observe that a thicker endometrium has an overt negative impact on ART outcome.101-104

Endometrial Echogenicity: a Biomarker for Progesterone Action Echogenicity reflects the different tissue interactions with ultrasound. Tissue echogenicity ranges from near sonotransparent as encountered in certain constituents of the body, notably water, to the highest echogenicity, produced by air (present in the bowel). On gray scale imaging, low echogenicity, as that of water, is commonly depicted in black, while high echogenicity appears in white. Typically, solid tissues with intermediate echogenic characteristics are depicted by degrees of gray with final grade depending on the water and/or air content and the number of such interphases. In the uterus, the echogenicity of the myometrium remains constant throughout the menstrual cycle and following various hormonal treatments. In contrast, the echogenicity of the endometrium varies between the follicular and luteal phases of the menstrual cycle and in response to exogenous hormones. In a prospective trial on 80 infertile patients, Forrest and colleagues105 showed that endometrial echogenicity increases after ovulation under the influence of progesterone. These authors were first to recognize the hypoechoic characteristics of the follicular phase endometrium (Fig. 35.4). The endometrium is characterized by a hypoechoic functional layer bordered by the hyperechoic basal layers with the center hyperechoic line produced by the interphase of the virtual endometrial cavity. Together, the hypoechoic functional layer of the endometrium and its outer limits produce the typical three-line pattern. Changes in endometrial echogenicity are seen soon after ovulation. The endometrium progressively acquires its hyperechoic features characteristic of the luteal phase, starting from the endometrium basalis layer and expanding upward. Forrest and colleagues105 observed a hyperechoic endometrium in 78% of women scanned during the luteal phase. Likewise, Templeton’s group identified a similar

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sequence of changes from hypoechogenic endometrium in the follicular phase to a primarily hyperechogenic pattern in the luteal phase of stimulated cycles.68 The sonographic changes from the follicular phase to those characteristic of the luteal phase start to be visible within 48 hours of ovulation. The hyperechoic transformation of the functional layer of the endometrium takes 4 to 7 days to complete. Grunfeld and colleagues studied the changes in endometrial echogenicity that took place in 18 women receiving sequential E2 (0.2 to 04 mg/day, transdermally) and progesterone (50 mg/day IM) treatment in preparation for donor egg IVF.106 Vaginal ultrasounds were performed starting before the onset of progesterone administration, and every 3 days thereafter until the 8th day of progesterone, when an endometrial biopsy was performed. Endometrial echogenicity was scored as pattern I, showing a triple-line pattern before the start of progesterone treatment, to pattern III, showing full hyperechoic transformation of the layer fucntionalis from the lamina basalis all the way to the endometrial lumen (Fig. 35.5). These authors concluded that endometrial thickness was a poor discriminator between advanced and retarded stromal changes on endometrial biopsies. Conversely, the degree of hyperechogenic changes on ultrasound (complete or partial) correlated with the degree of advancement of luteal changes (condensation, predecidualization) in the endometrial stroma. All women whose endometrial biopsy revealed delayed secretory changes in endometrial glands and stroma had ultrasounds showing partial hypoechogenic images (pattern II) (Fig. 35.6). Analyzing endometrial echogenicity on the day after hCG administration in ART, Gonen and Casper observed a typical hypoechogenic triple-line pattern in 49% (60/123) of subjects.107 In this subgroup of ART participants, the pregnancy rate was 30% (18/60), which was significantly higher than the figure obtained in the whole cohort (19.5%). Conversely, a full and partially hyperechogenic endometrium was found in 33% (41/123) and 18% (22/123) of women in whom pregnancy rates at 9% and 9.1%, respectively, were markedly lower than in their hypoechogenic counter parts. The original data of Casper and Gonen on the poor prognosis value of a hyperechoic endometrium in the late follicular phase of ART were largely confirmed in other publications,108-113 while others failed to find this relationship.114-115 In an effort to analyze how ultrasound data, namely, echogenicity, could reflect endometrial receptivity, we studied the endometrial appearance on ultrasound on the day of hCG administration in 228 consecutive COH cycles.116 We only included women younger than 38 years of age whose uteri were morphologically normal, and in a position that offered optimal visualization on ultrasound. Ultrasound images were digitized and analyzed using a computer-assisted system designed for measuring the degree of endometrial echogenicity (Fig. 35.7). Specifically, we studied the degree of the hyperechogenic changes that develop from the basal endometrium upward during the follicular-luteal transition, as described in E2 and progesterone cycles,106 but at times reported in ART before exposure to progesterone, at the time of hCG administration.107 Our results indicated that in the selected population of IVF candidates 34/228 (14%) had a fully hypoechoic triple-line endometrium (less than 30% hyperechoic at the base of

FIGURE 35.5  Hyperchogenic or type III endometrium: Under the influence of progesterone, the endometrium becomes hyperechogenic in relation to the surrounding myometrium during the luteal phase. From studies conducted in estradiol (E2) and progesterone substitution cycles, it has been determined that the endometrium becomes fully hyperechogenic, usually after 4 days of exposure to luteal phase levels of progesterone. The hyperechogenic characteristics of the secretory endometrium are believed to result from the abundance of mucus-filled tortuous glands, which generate many ultrasound interphases and, therefore, increase the overall echogenicity of the endometrium.

FIGURE 35.6  Intermediate or type II endometrium: During the first days of the luteal phase there is a characteristic thickening of the endometrial basalis, which progressively spreads upward to ultimately reach the hyperechogenic interphase of the virtual endometrial cavity. The condition encountered after 2 days of exposure to progesterone when the hyperechogenic endometrial basalis extends over approximately 50% of the whole endometrial layer is described as a type II endometrium.

the endometrium) while 28/228 (12%) had a fully hyperechoic endometrium (greater than 70% of hyperechoic invasion of the functionalis layer).116 The remaining 166 women had 4 degrees of progressive hyperechoic changes that extended from 31% to 40% to 61% to 70% of the full functionalis layer. Demographic, hormonal and biological characteristic of each of these groups were similar, including, endometrial thickness and plasma progesterone levels,

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Image digitization

Selection

Gray level analysis

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Results Hyperechogenicity

Hyperech. ant.: 19% Hyperech. post.: 15% Hyperech. mean: 17% End. thickness: 9.5 mm

Type I Endometrial basalis

Hyperechogenicity

Hyperech. ant.: 100% Hyperech. post.: 100% Hyperech. mean: 100% End. thickness: 8.6 mm

Type III Endometrial hyperechogenicity

FIGURE 35.7  Computer assisted measurement of endometrial echogenicity. Endometrial echogenicity was studied on transverse slices. In the follicular phase (type I endometrium), one recognizes the characteristic hyperechogenicity of the endometrial basalis and that generated by the virtual endometrial cavity, outlining the hypoechogenic functionalis. In the luteal phase (type III endometrium), the hyperechogenicity ­pattern spreads to ultimately reach the virtual endometrial cavity, producing the characteristic hyperechogenic appearance of the endometrium. (From Fanchin R, Righini C, Ayoubi JM, et al. New look at endometrial echogenicity: objective computer-assisted measurements predict endometrial receptivity in in vitro fertilization-embryo transfer. Fertil Steril 74:274, 2000.) 6 echogenicity groups Extent of hyperechogenic transformation Endometrial thickness

≤30% (n = 34)

31%-40% (n = 37)

41%-50% (n = 37)

51%-60% (n = 55)

61%-70% (n = 37)

>70% (n = 28)

FIGURE 35.8  Computer assisted endometrial echogenicity showing the extent of hyperechogenic transformation on the day of human chorionic gonadotropin (hCG) and pregnancy and implantation rates. The figure shows six echogenic groups of increasing endometrial echogenicity. Pregnancy and implantation rates (59%, 57%, 35%, 20%, 16%, and 11%, respectively) and implantation rates declined from 59% to 11% and 35% to 3%, respectively when hyperechogenicity ranged from <30% to >70% of endometrial thickness. (From Fanchin R, Righini C, Ayoubi JM, et al. New look at endometrial echogenicity: objective computer-assisted measurements predict endometrial receptivity in in vitro fertilization-embryo transfer. Fertil Steril 74:274, 2000.)

which remained less than 1 ng/mL in each group. This latter observation indicates that the premature increase in echogenicity seen in certain IVF patients is not the result of premature luteinization with increased progesterone production. Finally, the number of embryos transferred was also similar in all six groups; in each case two to four embryos were transferred (as commonly done in the 1990s). Pregnancy and embryo implantation rates varied greatly, however, between the different echogenicity-based groups. From a highest value of 59% and 16%, respectively, for the hypoechogenic group, it decreased to 23% and 3%, respectively, for the hyperechoic group, with progressively diminishing results for increasing echogenicity in the intermediate groups (Fig. 35.8). In a different study, we assessed endometrial echogenicity on the day of hCG administration, oocyte retrieval, and embryo transfer (day 2) in patients whose plasma progesterone was below or above the cut off value of 0.9 ng/mL.117 On the day of hCG, the mean hyperechoic transformation of the functionalis layer of the endometrium was similar in the two

groups at 40% and 41%, respectively. During the 4 days that followed hCG administration, the hyperechoic transformation was much faster, however, in the high-progesterone group, with values of 70% versus 63% at oocyte retrieval and 90% versus 79% at the time of embryo transfer (ET), respectively. The histological basis for the premature hyperechogenic transformation seen in certain IVF women remains unclear. During the follicular-luteal transition, it has been hypothesized that the development of spiral arteries and the coiling of the glands which takes place under the influence of progesterone causes the increase in echogenicity. The fact that progesterone was low (less than 1 ng/mL) in women showing premature echogenicity excluded this simple explanation. Work from Devroey’s team performing endometrial biopsies on the day of oocyte retrieval in the cycle of embryo transfer confirmed that premature lutenization did not explain the early hyperechogenic transformation.118,119 This leaves us with no explanation for the increase in echogenicity found in certain women on the day of hCG

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administration in ART, an observation bearing ominous predictive value for ART outcome. There are considerable inter-study differences in the predictive value of various echogenic patterns on the day of hCG, oocyte retrieval and embryo transfer for which no explanations are readily available.120 We believe that some of the variability in the ultrasound patterns reported stems from methodological issues. For example, the endometrium of women whose uteri are in an intermediate position is likely to erroneously appear hyperechoic due to the fact that the ultrasound beam tends to hit endometrial glands at an angle rather than being parallel with the glands when the uterus is in a marked anteverted or retroverted position. In an unpublished trial, we performed manual rotations of freshly removed uteri before an ultrasound probing. This confirmed that the hypoechoic pattern obtained when the uterus is held in the anteverted or retroverted position before the probe becomes hyperechoic when the intermediate position is mimicked.73 Recently, Dietterich and colleagues indicated that transabdominal screening likely limits the possibility of false interpetation.101

Three-Dimensional Volume Reconstruction of the Endometrium Interest in measuring endometrial volume rather than its thickness is based on obtaining a more global parameter for assessing endometrial priming by estrogens.11 Progress made in 3D technology allows the subendometrial junctional zone to be assessed.121 Early assessment of endometrial volume estimated from 2D ultrasound images by simple formulae such as the prolate ellipsoid122,123 were of little help for studying the uterus, probably because of the irregular shapes of this organ which made volume calculation based on theoretical formulas inaccurate. Off-line and later, built-in 3D volume reconstruction systems incorporated in the ultrasound machine offer supperior approaches for ultrasound-based volume measurements. 3D-based volume calculation of territories such as the endometrium use either of the two following methods: (1) The simplest or multiplanar approach consists of scrolling through multiple serial slices, which allows more measurements of irreguarly shaped objects.124 (2) A more elaborate approach is based on using a virtual organ computer-aided analysis imaging program (VOCAL) developed by a 3D ultrasound manufacturer, GE-Kretz, Zipf, Austria. A detailed description is provided by Raine-Fenning and colleagues.12 Briefly, 3D reconstruction encompasses two primary steps. First, the relevant boundaries of the volume to be studied are identified on 2D gray-scale images in order to delineate the limits of the 3D reconstruction. For the endometrium volume, the endometrial to myometrial interface is either manually, or semi-automatically identified by the examiner on 2D images, using the B (transverse) or C (coronal) plane. Second, the 3D volume is rotated about a central axis through sets of user-defined steps ranging from 6 degrees to 30 degrees at a time. It takes from 30 to 6 planes, depending on the rotating pattern chosen, to complete a full 180-degree rotation. Finally, volume acquisition is generated automatically upon completion of a full 180-degree rotation (Fig. 35.9A-C). Experimental calculations indicated that the volume is measured with increasing precision with the

increased number of rotation steps.125 Practically, 9-degree rotations leading to a total of 15-degree rotation steps73 are preferred. From a first volume of reference (i.e., the endometrial volume), it is possible to generate secondary volumes that extend to set distances of the primary reference. For example, 1 to 5 mm outer extensions of the primary endometrium volume, using the shelling properties of the VOCAL system, can define subendometrium volumes. In a prospective trial, Raine-Fenning and colleagues scanned a population of 30 presumably fertile volunteers on alternate days until evidence of ovulation was obtained and collapse of the follicle was witnessed, and every 4 days thereafter.126 3D volumes of the uterus were acquired using a Voluson 530D ultrasound machine from an appropriate longitudinal scan of the uterus. 3D volume measurements were performed off-line using the VOCAL system with endometrium boundary identification conducted on the coronal C plane in successive manual 9-degree rotation steps. As illustrated in Figure 35.9B, the changes in endometrial thickness and volume throughout the menstrual cycle followed similar patterns with progressive increments during the follicular phase and stable values throughout the luteal phase. The changes in endometrial thickness and volume were highly correlated (R2 = 0.767; P < 0.001). Studies looking at correlations between endometrial volume and ART outcome revealed the same kind of relationship already described for endometrial thickness. Like their endometrial thickness counterparts, 3D endometrial volume studies revealed poor pregnancy rates in cases of marginal endometrial volume. Specifically, pregnancy rates were directly related to endometrial volume for values from 2 to 4 mL, significantly reduced when the endometrium was less than 2 mL, with no pregnancy if less than 1 mL. Conversely, further increases in endometrial volume greater than 4 mL were not associated with better outcomes, as compared to 2 to 4 mL.11 In a study of 47 women, Schild and colleagues showed no correlation between endometrial volume and pregnancy rates,124 as did Yaman and colleagues.127

Uterine Doppler Principles and Applications The Doppler effect (named after the German physicist, Christian Doppler) refers to the phenomenon whereby an apparent change in frequency is perceived when relative motion exists between the wave source (in our case, the reflected ultrasounds) and the receiver. Yet, the emitted frequency (here, the ultrasounds refected to the receiver) does not change. The frequency change is only perceived by the receiver, as the source-receiver distance changes due to movement. If ultrasounds are emitted (or reflected) from a source that moves toward the receiver, an increase in frequency will be perceived. The reverse will be perceived if the ultrasound source moves away from the receiver. In both cases, the perceived frequency change (i.e., the intensity of Doppler effect) is proportional to the speed of displacement of the target on which ultrasounds are reflected. The primary application of the Doppler effect has been for analyzing the movement of blood particles on which ultrasounds are reflected for studying blood flow. Depending on the methodological approach used, Doppler-based

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A

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B

C FIGURE 35.9  Endometrial volume calculation using a virtual organ computer-aided analysis (VOCAL) imaging program also allows the calculation of a secondary subendometrial volume extending at 5 mm (A and B) or 1 mm (C) outside from the endometrial myometrial interphase.

analyses may (1) look at individual vessels or; (2) measure perfusion in a whole tissue area. The former approach assessing flow in specific vessels (i.e., the uterine artery) rely on the pulsed-Doppler technique. This consists of sending sound signals at timed intervals in a defined volume or sampling gate, and studying the perceived frequency changes of the return signal or Doppler waveform. Doppler-based blood flow analyses stem from advances in cardiology, which established the methodological foundation for all Doppler analyses performed today.128 Pulse Doppler (Fig. 35.10) involves first physically locating the vessel to be studied in order to properly position the Doppler sampling gate. This can be done using a duplex system, which provides concomitant gray scale or color Doppler imaging for vessel identification (see later in the chapter). Once the emitting signal is targeted at the designated vessel, blood flow and resistance are calculated by analyzing the frequency changes perceived or Doppler waveform. The intensity of the Doppler effect reflects the speed of displacement of the blood particles toward or away from the probe, which emits and receives the return signal. An exact computation of blood flow and resistance implies, however, that the angle between the ultrasound wave and the great axis of the vessel is known. This is needed for determining the true displacement speed of the blood-borne particles.

FIGURE 35.10  Pulsed Doppler waveform analyzed by computation of the semiquantitative pulsatility index (PI), which reflects the impedance to flow in the territory downstream from the area of measurement.

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This calculation is, unfortunately, impossible in gynecological organs because uterine and ovarian vessels are far too tortuous. The pulsed-Doppler technology in gynecology must, therefore, rely on approximations for assessing the Doppler signal, as an accurate determination of blood flow and resistance are impossible.9,10,129,130 Practically, the impedance to flow, a reflection of vascular resistance, is assessed by descriptive analysis of how the Doppler flow wave is modulated through the systolic and diastolic phases of the cardiac cycle. This permits semiquantitative measurements of systolic and diastolic signal ratios, or Doppler indices. The most common of these indices are the resistance indices (RI) and pulsatility indices (PI). RI (values from 0 to 1) and PI (values from 0 upward) are directly related to vascular resistance, with higher values corresponding to lower flow. The alternate approach for Doppler analysis uses more complex real-time assessment of the Doppler effect directly on the return ultrasound signal serving gray scale imaging. This approach applies color-coding to voxels that are subjected to Doppler effect, while the rest of the voxels serve for gray scale imaging. The end result or color Doppler function and its variant angle-independent power Doppler, provide direct imaging (or mapping) of vascular flow overlying gray-scale ultrasound images. Color and power Doppler, therefore, offer a novel way for assessing vascularity in a given organ or territory. Color and power Doppler also permit global assessment of vascularization in a given area by direct computer-based visual analysis of colored voxels present over the total number of voxels, or power Doppler angiography (PDA).87 Unlike PI and RI, PDA values are directly related to local flow, with higher values indicating higher flow. Various systems have been developed for filtering nonspecific color Doppler signals or “flash” artifacts that are triggered by displacements of the targeted tissue as a result of bowel or respiration-linked movements. More recently, computer-assisted power Doppler assessments have been coupled with 3D volume reconstruction (3D-PDA) for automated operator-independent measurement of vascularity in a precisely defined volume.131 (Fig. 35.11A and B). This approach offers improved reproducibility, because measurements are conducted in an anatomically and electronically defined territory, such as the endometrium or ovary.12 3D-PDA is measured in the volume of interest previously defined using VOCAL.12 The results of 3D-PDA are displayed on a histogram from which three indices, the vascular (VI), flow (FI), and vascular/flow indices (VFI), are derived. VI (%) represents the proportion of lighted voxels over all voxels, thus reflecting the degree of vascularity of the studied volume. FI (0-100) represents the mean power Doppler intensity of the lighted voxels, a parameter meant to reflect the vascularization flow rate. VFI (0-100) is a product of the two indices VI and FI (Fig. 35.12). The Doppler function of ultrasound makes it possible to extend the diagnostic usefulness of gray-scale imaging. Doppler data have been used for two main purposes: (1) refining cancer detection by identifying or excluding suspicious vascularitiy in relation with gray-scale findings (i.e., an ovarian cyst); (2) assessing the effects of hormones (endogenous or exogenous) on blood flow in an attempt to identify markers of endometrial receptivity.

A

B FIGURE 35.11  Computation of color Doppler finding in a given area by computation power Doppler angiography (PDA) in a 3D defined territory, as for example, the endometrium and subendometrial volume using the virtual organ computer-aided analysis (VOCAL) and shelling functions. PDA score is directly related to flow. Three primary indices are computed, the vascularization (VI), flow (FI), and vascularization-flow index (VFI). VI reflects the number of vessels in a given area whereas FI is proportional to blood flow in these vessels and VFI constitutes a product of VI and FI. A, In normal conditions, color Doppler shows no to minimal vascular entries in the endometrium. B, Endometrial polyp vessels are shown entering inside a given territory of the endometrium (right cornua).

Vascular Changes Related to the Menstrual Cycle and Hormonal Effects Investigators have tried to follow the changes in uterine vascularization that are tied to hormonal changes in the menstrual cycle. Early studies using pulsed Doppler looked at changes in Doppler waveforms recorded from the uterine artery9 and its major branches.129,132,133 Later, Raine-Fenning and colleagues used 3D-PDA to quantify changes taking place in endometrial and subendometrial blood flow during the menstrual cycle.126 These authors observed that both endometrial and subendometrial blood flow increased during the follicular phase, peaking 3 days prior to ovulation, and decreased to a nadir 5 days post-ovulation (Fig. 35.13). This pattern differs, however, from prior findings made with pulsed Doppler showing a steady increase of blood flow throughout

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Contour (shell) - Histogram Gray

Color Angio

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MG (0, 100) 35.153

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Mean gray value Vascularization index Flow index Vascularization flow index

(%) 10.276 (0, 100) 28.000 (0, 100) 2.877

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Return

the menstrual cycle.126,129,134-137 In Raine-Fenning’s trial, smoking was associated with significantly lower VI and VFI throughout the menstrual cycle, whereas parous women had higher values than their nulliparus counterparts.126 Exogenous hormones administered to women suffering from premature10,133 or normally occurring menopause132 induced a prompt and profound decrease in uterine PI and RI indices. This was interpreted as reflecting the vasodilatory effects of E2. Menopausal138 and donor-egg IVF regimens10 were associated with similar vasodilation of uterine arteries, suggesting that maximal vasodilation is achieved at levels of E2 found within menopause treatments. Conversely, exposure to higher E2 doses as used in donor-egg regimens caused no further increase in flow. In these studies, the vasodilation of uterine arteries induced by E2 administration was partially antagonized by synthetic progestins,138 but not by progesterone administration.10 Vascular changes observed in the uterus during COH do not parallel those that would be anticipated from the mere increased exposure to E2. These findings indicate that the stimulated ovaries produce vasoactive factors other than E2. In a prospective trial, Ng and colleagues138a compared endometrial and subendometrial blood flow measured using the 3D-PDA approach in menstrual and COH cycles of the same women. Their findings showed a significant decrease of endometrial and subendometrial blood flow during the stimulated cycle,138a with excessive responders having further reductions in endometrial blood flow as compared to moderate responders.138a These findings suggest that vasoconstrictive substances are released by the stimulated ovaries that antagonize the vasodilatory properties of E2.88,139 Among the putative factors possibly responsible for the decrease in uterine flow during COH are ovarian androgens, which are produced in increased amounts under the influence of exogenous follicle-stimulating hormone (FSH).53,140 Supporting the concept that ovarian androgens interfere with the vasodilatory properties of E2 is the observation of increased uterine artery resistance in case of polycystic ovary syndrome (PCOS) made by some,141 but not all investigators.142 Further supporting the vasoconstrictive role of androgens, or of a substance released in conjunction with androgens, is the report of an improvement of uterine flow in PCOS following administration of the antiandrogen,

FIGURE 35.12  Computer assisted analysis of “lighted” voxels in a given territory leads to the computation of two distinct indices, the vascular (VI) and flow indices (FI), with VFI being of a product of both. VI represents the number of vessels in a given territory whereas FI is proportional to the intensity of blood flow in these vessels.

flutamide.141 In a different study, Ng and colleagues142a observed a negative correlation between E2 levels and blood flow suggesting that the putative vasoactive factor produced during COH is triggered by FSH administration in a proportional manner to that of E2. This is further supported by the observation that the decrease in endometrial flow is more pronounced in women whose response to COH is excessive as compared to moderate responders.142b The promising findings of early Doppler studies indicating that Doppler values predicted endometrial receptivity143 have not been confirmed by recent 3D-PDA data from either fresh5 or frozen embryo transfer cycles.5 Yet, increased vascularitiy may be predictive of lesser risk of miscarriage.82

Dysfunctional Uterine Bleeding: Benign and Malignant Endometrial Pathology Endometrial pathology, such as polyps or submucosal fibroids, are sought in cases of: (1) dysfunctional uterine bleeding (DUB) or; (2) as part of an infertility workup. Odeh and colleagues prospectively evaluated DUB in 89 perimenopausal and 56 menopausal women.144 In their hands, endometrial volume measurement more accurately predicted endometrial hyperplasia and/or carcinoma than simple endometrial thickness measurement. Receiver Operating Characteristics (ROC) curve analysis indicated that a sensitivity of 100% with a cut off value of 1.45 mL, which resulted in low sensitivity of 0.08%. A cut off value of 3.56 mL for the endometrium volume offered a sensitivity of 93% and a specificity of 36%. With this value, two carcinomas would have been missed in the patient cohort; one with alarming 3D-PDA data, the other with negative 3D-PDA results. Consequently, new tools such as 3D-based volume measurements should not cause us to ignore careful analysis of the endometrium. Irrespective of endometrial thickness or volume values, a heterogeneous echogenicity with close juxtaposition of hyperechoic and hypoechoic aspects warrants histological exploration. When endometrial polyps are suspected based on an irregular endometrium on plain ultrasound, direct power Doppler may identify the feeding vessel entering the polyp. Conversely, endometrial vessels are normally not viewed on power Doppler.145 Suspicious findings on plain ultrasound justify a contrast-enhanced study.

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7 Vascularization index (%)

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FIGURE 35.13  Temporal variation of three-dimensionalpower Doppler angiography (3D-PDA) vascularization index (VI), flow index (FI), and vascularization flow index (VFI) throughout the menstrual cycle in endometrial and subendometrial territories. A characteristic postovulatory decrease in blood flow is observed in both endometrial and subendometrial territories. (Raine-Fenning NJ, Campbell BK, Kendall NR, et  al. Quantifying the changes in endometrial vascularity throughout the normal menstrual cycle with three-dimensional power Doppler angiography. Hum Reprod 19:330–338, 2004.)

Vascularization flow index

3 2.5 2 1.5 1 0.5

The cardinal sign of endometrial carcinoma is an abnormally thick endometrium,146,147 often with irregular and erratic echogenicity.148 In 339 menopausal women presenting with DUB, there was no endometrial cancer developing over a 10-year follow-up with an endometrial thickness of ≤4 mm.149 A more recent meta-analysis concluded that earlier studies probably overestimated the diagnostic accuracy of endometrial thickness.150 A more conservative cutoff level of 3 mm was recommended for exclusion of endometrial carcinoma in women with postmenopausal bleeding.150 Doppler has been claimed to differentiate between cancer and noncancerous lesions. While noncancerous lesions tend to have higher Doppler indices, there is an overlap between

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the PI and RI values of benign and malignant lesions.151 Based on these findings, Tabor and colleagues concluded that postmenopausal bleeding requires an endometrial sampling irrespective of the thickness measured on ultrasounds.152 Finally, in cases of endometrial cancer, vaginal ultrasound and MRI were equivalent for determining the depth of myometrial invasion.153

The Myometrium: Physiological and Pathological Findings Uterine leiomyomas or fibroids are the most common solid tumor, occurring in 20% to 40% of women in their

CHAPTER 35  Pelvic Imaging in Reproductive Endocrinology

FIGURE 35.14  Uterine fibroid. Characteristically, vessels run around the surface of the fibroid from which radiating branches penetrate vertically into the tumor.

reproductive life.154 Fibroids are well-circumscribed lesions, often with a pseudo capsule containing feeding vessels running at the surface of the fibroid, which penetrate into the fibroid in a radiating manner (Fig. 35.14). This pattern differs from that of adenomyosis, in which vessels are sparse and scattered, and the borders of the lesion typically indistinct. Fibroids originate from the middle layer of the myometrium from which they tend to expand either inward toward the cavity to become submucosal or outward toward the serosa, possibly to the point of becoming pedunculated. The possibility that fibroids may cause infertility and constitute a predisposing factor for miscarriages depends primarily on their location and degree of extension. First, fibroids may alter fertility by interfering with sperm transport and embryo implantation.155 This detrimental effect of fibroids on sperm transport has recently been supported by MRI-based studies of uterine contractility.156,157 Kido and colleagues observed that submucosal fibroids were more likely to affect uterine peristalsis than their intramural counterparts.157 Second, the possibility that fibroids alter endometrial receptivity has been entertained for many years. Fibroids may hamper embryo implantation, in part by physical effects when present in a subendometrial location, in part by focal endometrial disturbances158 and local inflammation through the release of vasoactive and other substances.159 The possibility that fibroids cause miscarriages remains a subject of debate. In a recent study, miscarriages were more frequent in a population that experienced fibroids than in their unaffected counterparts.160 There are many other studies, however, that failed to find such an association.161 There are new therapeutic avenues for treating fibroids medically using selective progesterone receptor modulators (SPRMs).162 However, the role of SPRMs in infertility treatment remains to be explored.

Contrast-Enhanced Imaging of the Uterine Cavity History, Principle and Practical Issues Early in the use of vaginal ultrasound it became apparent that uterine exploration in early pregnancy offered images

863

of the developing embryo of outstanding quality.163,164 The remarkable definition of fetal images obtained in early pregnancy using vaginal ultrasound is crucial for early detection of fetal malformations.165-167 However, ultrasound images of the nonpregnant uterine cavity yield lower quality because of the lack of intrauterine fluid playing the role of contrast enhancer.15 This led to the design of a new diagnostic procedure in which 0.9% NaCl or similar products are instilled in the uterine cavity for improving image quality. This procedure has been called hystero salpingo-contrast-sonography (HyCoSy), saline infusion sonography (SIS), or hysterosonography (HySo) (Fig. 35.15).15,17,18,168,169 Two types of contrast products provide either negative (black) or positive (white) contrast enhancement of the uterine cavity on grey scale imaging.170 Negative contrast solutions have the acoustic properties of water (sonotransparent) with normal saline being the most commonly used agent. Positive contrast solutions consist of preparations that gain sonorefracting properties from air entrapped in various microparticle systems from which it is progressively released over time.171-173 This is achieved through albumin (Albumex®) or saccharide microparticles (Echovist® later replaced by Levovist®, BR1, EchoGen, etc.). These positive ultrasound contrast products are injectable solutions available for various medical indications, but are used intrauterinely for gynecological imaging.174 Positive contrast solutions instilled in the uterine cavity provide white contrast enhancement of the uterine cavity. Unfortunately, this often obscures vision of the far-sided uterine wall because of shadowing effects created by the contrast product in the uterine cavity.175 Negative contrast solutions, such as normal saline, provide black contrast enhancement of the uterine cavity. This leaves the vision of the far side of the uterus totally unobstructed which is better for identifying intracavitary lesions.173,176,177 From these findings, it was rapidly recognized that negative contrast using saline is the preferred option for exploring the uterine cavity, whereas, positive contrast solutions are best for visualizing the fallopian tubes, organs that evade scrutiny on ultrasound examinatiton.178 Positive echographic contrast solutions have also been employed for enhancing the Doppler signal and improving the assessment of tissue perfusion when administered systemically.179 Yet, Doppler enhancement that requires IV administration of these contrast products has not yet provided clinically relevant advantages that would justify their use for diagnostic purposes. Lastly, a variety of harmonic imaging techniques with and without pulse sequencing methods have been developed for improving the signal-tonoise relationship.180,181 The approaches currently in development in various domains of ultrasonography may generate practical applications in gynecological assessment in the future.182 The relative complexity of the contrast-enhanced procedures, which requires the help of an assistant, limits the use of these methods, despite the quality of the images obtained and the reliability of the diagnoses.169,183 Two options exist for assuring the proper distension of the uterine cavity during the procedure: (1) A simple catheter of the type used for intra-uterine insemination (IUI) or one equipped with a specially designed cone-shaped stopper for better fit against

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A

C

B FIGURE 35.15  Hysterosonography (HySo): Intrauterine infusion of a contrast solution improves vision of the uterine cavity provided by plain ultrasound (A). Provides an interphase that enhances contrast (B). Facilitates the detection of intrauterine pathologies such as polyps or fibroids (C).

the external os of the cervix.8 (2) A balloon-equipped catheter, which is inflated after positioning in the uterine cavity in order to prevent displacement. With the former, the speculum needs to be removed very carefully after placement of the catheter in order to not displace it from its proper intrauterine position. If the catheter is not firmly held in place, it most often justifies using a speculum that is articulated on one side only, which facilitates extraction, but is notoriously uncomfortable for the patient. The balloon catheter is itself capable of creating some degree of discomfort when it expands in the lower uterine segment. Moreover, it may hinder visualization of pathology lying in the lower uterine segment. In short, albeit providing images of remarkable resolution, HySo is a three-hand procedure (one for holding the probe, one for instilling medium, and the third for making image adjustments and measurements). HySo, therefore, most often requires the help of an assistant. Recently, an innovative option has been proposed in which a phase shifting medium is used.168 This medium has a gel consistency at room temperature and the acoustic characteristics of water. Intrauterine instillation of 1 to 3 mL creates a slight distension of the cavity, thus offering negative or black contrast vision during the time necessary for ultrasound examination, which is conducted in the absence of any instrument present in the uterus. The medium later liquefies and is expelled naturally. In a pilot trial, this novel technique provided classical HySo-like images.184

Hysterosonography for Visualizing Endometrial Polyps and Fibroids Using HySo for visualizing intrauterine pathologies, as in cases of DUB, has advantages and pitfalls that have been recognized.185,186 In these women, HySo is in direct competition with other noninvasive diagnostic procedures such as diagnostic hysteroscopy.187 In a prospective trial, de Kroon and colleagues188 reported that HySo provided conclusive information in 84% of 180 consecutive women presenting with DUB. In all these cases, the positive and negative findings of HySo were confirmed in women undergoing surgery, for a sensitivity and specificity of 100%. In 5.6% of cases, however, HySo could not be performed because of cervical stenosis and its results were judged inconclusive in a further 10.3%. A meta-analysis of trials looking at the value of HySo in women with DUB reported pooled sensitivity and specificity of HySo in the 24 retained trials of 0.95 (95% CI: 0.93, 0.97) and 0.88 (95% CI: 0.85, 0.92), respectively.188 These results were confirmed by further trials and meta-analyses.189,190 Furthermore, HySo-targeted endometrial biopsies have been described that allow direct endometrial sampling in the area of interest.191 A survey found, however, that patients generally preferred office hysteroscopy over HySo.192 Concomitent use of 3D-reconstruction of uterine imaging further enhances the clinical value of HySo.193 3D volume acquisition allows for limited duration of uterine

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FIGURE 35.16  Three-dimensionalhysterosonography (3D-HySo) allows secondary review of images for de­tailed analysis of intrauterine contents.

distension to the time necessary for carrying out the electronic acquisition of a 3D volume. Furthermore, 3D reconstruction permits review of HySo images after uterine distension has subsided (Fig. 35.16). HySo can also benefit from the added information provided by Doppler, such as the detection of a vessel entering into a polyp or encircling the surface of a submucosal fibroid.193 Unusual uses of Doppler in conjunction with HySo includes the identification of an uteroperitoneal fistula194 and intrauterine vascular malformation.195 Besides DUB, other indications for HySo include evaluation of women receiving tamoxifen treatment,196,197 and according to some authors, evaluation of asymptomatic women entering menopause and considering hormonal therapy.198 Discomfort generated by HySo examination is usually mild to minimal. In a prospective trial, Dessole and colleagues199 looked at the side effects and complications of 1153 HySo procedures. In 93% of cases, HySo was successful. Side effects of moderate to severe pain, vaginal reactions, and nausea and vomiting occurred in 102 (8.8%) of cases. Severe complications such as fever and clinical signs of peritonitis were encountered in 0.95% of cases, with tubo-ovarian absesses having been reported.200 We do not routinely perform cervical cultures or use routine antibiotics; a recommendation shared by the Amercan College of Obstetricians and Gynecologists (ACOG).201 The risk of propagating cancer in case of endometrial cancer has been examined.202 Fluid spilling from the fallopian tubes was collected while HySo was conducted preoperatively in 32 women diagnosed with endometrial cancer. Malignant and suspicious cells were recovered from tubal spills in 2 (6.25%) and 6 (18.8%), respectively, for a total of 8 (25%) findings of suspicious or malignant cells recovered from the 32 subjects. These authors strongly recommended

that HySo not be performed in case of proven or suspected endometrial cancer.202 The value of routine pre-ART screening of the uterine cavity using hysteroscopy or HySO is being discussed.203-205 In our program, women are synchronized with a short 7- to 14-day course of oral contraceptive (OC) pills during which time routine HySo is performed. We commonly remove incidental polyps while the patient is still on the OC pill. Performing HySo while the patient is on the pill offers the advantage of having an atrophic mucosa which precludes confusing a mucosal fold for a polyp. HySo or hysteroscopy should be part of the regular workup in case of repeated miscarriages.206 It is important to underscore that a comparison between HySo and office hysteroscopy goes beyond the ability to detect polyps, as each technique offers respective advantages. On the one hand, office hysteroscopy provides information about possible chronic inflammation or infection of the endometrium not otherwise identifiable,88 including by cervical cultures.207 On the other, HySo combined with pelvic ultrasound offers information about organs other than the uterus, namely the ovaries and possible cysts, that may be pertinent for sorting out the cause of DUB. Hence, the choice between HySo and office hysteroscopy should result from a process that takes into account all the issues pertinent to each clinical situation.

Hysterosonography in ART In light of ART costs, many recommend routine assessment of the uterine cavity for the presence of polyps prior to ART using either HySo or office hysteroscopy, which are equally effective.208,209 A recent meta-analysis reported data that collectively supports the value of removing even small polyps.210 Hysteroscopic removal of endometrial polyps of 16 mm (mean) detected by ultrasound doubles the pregnancy

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rate in patients undergoing intrauterine insemination.210 In patients with two failed ART cycles, diagnostic or operative hysteroscopy before a subsequent attempt appears to improve outcomes.210,211 It should be noted, however, that such benefit may result from nonspecific effects of any type of endometrial injury.212 Fearing that systematic pre-ART HySo may lead to false positive findings generated by mucosal folds, it is recommended that HySo be performed either in the first 10 days of the follicular phase or better while on OC pill.

Müllerian Anomalies and Recurrent Pregnancy Loss The true prevalence of uterine anomalies is uncertain because of the disparate reported numbers, with incidence ranging from 0.16% to 10%,213 mainly based on interpretation of hysterosalpingograms. A selection of better structured studies reported a similar incidence of approximately 1% in infertile or presumably fertile women.214-216 A higher incidence, approximating 3%, was found, however, in women having a history of recurrent pregnancy loss.217-219 Early in development (approximately 9 to 10 weeks), the common understanding has been that paramesonephric ducts fuse caudally while the uterine septum starts regressing in an unidirectional manner from the lower part of the uterocervical canal upward.220 An alternate bidirectional regression theory exists, however.221 In a recent study, Mazouni and colleagues undertook a full evaluation of 110 women suspected of having a müllerian anomaly, using vaginal ultrasound, hysterosalpingography (HSG), computarized tomography (CT) scans, and in cases diagnosed in the later years, MRI.222 Müllerian duct anomalies were reported according to the American Fertility Society (AFS) classification (Fig. 35.17).223 Of the 110 patients referred for müllerian anomalies, 73 women had a septate uterus, 20 a bicornuate uterus, 10 uterine hypoplasia, 4 a unicornuate uterus, and 3 Mayer-Rokitansky-KusterHauser syndrome. In 33% of the cases, the diagnosis was made as part of the infertility work-up, in 18.2% because of repeat early spontaneous abortion, and in 12.7% on the occasion of ultrasound performed during pregnancy. In cases of septate vagina, dyspareunia was a presenting symptom, accounting for 8.2% of all müllerian anomalies in this series. The issue of how müllerian anomalies are best diagnosed has not yet been resolved. HSG was the traditional approach, and remains a useful if not indispensable examination in many cases. MRI, although helpful, should not be looked at as the primary means for diagnosing müllerian malformations, but rather used secondarily for defining cases too complex or atypical for definitive diagnosis on ultrasound.222 3D ultrasound with contrast enhancing solution stands as the diagnostic tool of choice for first-line screening for uterine malformations. Yet, admittedly, this novel approach has not been sufficiently challenged by use in low-prevalence populations. However, Woelfer and colleagues prospectively studied 1089 women with no history of infertility or recurrent early fetal losses using a 3D ultrasound.224 The scanned uteri were analyzed in reconstructed coronal planes, looking at a series of parallel transverse sections and analyzed according to the AFS classification (Fig. 35.17). Briefly, normal and arcuate uteri were recognized by their convex external contour with indentation less than

10 mm. The fundal contour, which was straight or convex in the normal uterus, was concave in the case of arcuate uterus (indentation at obtuse angle) and showed the presence of a septum with the central point of the septum at an acute angle. In the bicornuate uterus, the external contour showed an indentation greater than 10 mm dividing the two cornua with a convex fundal contour in each. Of the original 1289 women recruited into the study, 200 were not suitable for analysis because of poorly visualized uterus primarily due to fibroids distorting the cavity. Of the remaining 1089 women, 983 had a normally shaped uterine cavity. Of the 106 (9.7%) uterine anomalies, there were 72 (68%) arcuate, 29 (27%) septate, and 5 (4.7%) bicornuate uteri.211 Extending their research on uterine malformations, this team looked at uterine anomalies in 509 women with a history of unexplained recurrent miscarriages and 1976 asymptomatic women in whom a uterine anomaly had been diagnosed fortuitously on ultrasound.225 There was no difference in relative frequency of various müllerian anomalies or depth of fundal distortion between the two groups. However, in the case of both arcuate and septate uterus, the length of the remaing undisturbed uterine cavity was significantly shorter with more pronounced distortion in the recurrent miscarriage group. This observation is clinically important. It indicates that it is the degree of the müllerian malformation that bears the most important consequences on reproductive outcome rather than the type of malformation encountered.

Uterine Contractility: Direct Visualization on Ultrasound Contractility of the Nonpregnant Uterus: Methodological Issues Detection of contractions in the nonpregnant uterus previously required the invasive measurement of intra-uterine pressure (IUP). The ability to study uterine contractility by ultrasound has revived interest in this facet of uterine physiology, leading to multiple studies aimed at understanding the role of contractility in reproductive physiology. Uterine contractions (UC) are characterized by three parameters: frequency, amplitude, and direction of contraction. Also pertinent to the contractile status of the uterus is the IUP value that prevails between UCs or resting tone. Identifying UC on ultrasound scans has benefited from various forms of image management, which were necessary because of the relatively slow frequency of UC (maximum of 5 UC per minute in the late follicular phase), making direct UC identification difficult. Most commonly, uterine scans are accelerated by 5 to 10 times for UC identification, using fast plays of VHS recordings226,227 or digitized image sequences.83 Some investigators have used custom computer assisted systems for UC recognition and computation of UC frequency. Ayoubi and colleagues83 used an off-line 3D reconstruction system providing time mode image sequences depicting time-related changes of the myometrial to endometrial interphase (Fig. 35.18). On these sequences, UCs appear as successive waves created by the periodic displacement of the myometrial to endometrial interphase. We demonstrated in a prospective trial that the 3D-based approach and direct identification of UC on fast plays of digitized image sequences provide identical

CHAPTER 35  Pelvic Imaging in Reproductive Endocrinology

I Hypoplasia/agenesis

(a) Vaginal

(c) Fundal

II Unicornuate

(b) Cervical

(d) Tubal (e) Combined

III Didelphus

(a) Communicating

(b) Noncommunicating

(c) No cavity

(d) No horn

V Septate

IV Bicornuate

(a) Complete

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(a) Complete

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VII DES drug related

(b) Partial

A 3 1

2

3 1

3

1

2

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3

B FIGURE 35.17  Müllerian anomalies: A, American Fertility Society classifiaction of müllerian anomalies. B, differentiation of septate from bicornuate uteri. The uterus is bicornuate when apex of the fundal external contour occurs below (1) or within 5 mm above (2) a straight line between the tubal ostia. The uterus is septate when apex is more than 5 mm above the line between the tubal ostia (3). DES, diethylstilbestrol.

UC frequency results.83 More recently, Pierzynski and colleagues used a system based on image distortion recognition for UC identification.228 In a prospective trial conducted in estrogenized women, Bulletti and colleagues reported perfect concordance between UC frequency measurements made by IUP analysis and UC recognition on ultrasound scans using a 3D-based approach.229 This study provided definitive validation of the noninvasive ultrasound-based approach for UC frequency measurement. The direction of contractions has been assessed by visualizing the displacement of the contractile process on fast plays of uterine scans. Based on direct UC visualization, various types of contractions have been described, ultimately leading to the recognition of two primary types: either retrograde, cervix to fundus; or antegrade, fundus to cervix. It

is of note, however, that ultrasound-based analysis of UC displacement have never been validated by comparing ultrasound findings against the actual propagation of the pressure wave, as measured by multiple IUP recorders230 or displacement of the uterine contents.231 UC amplitude and uterine resting tone also evade ultrasound-based analyses.157,232

Uterine Contractility During the Menstrual Cycle: Three Characteristic Patterns There are typically three patterns of uterine contractility that are recognized during the menstrual cycle. These are emblematic of the late follicular phase, luteal phase, and intercycle interval (i.e., during menses).83,233 Each of these patterns is defined by sets of UC characteristic such as frequency, amplitude, and direction of contractions.

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FIGURE 35.18  3D-based approach for uterine contraction (UC) assessment. 2D images are recorded over time without sweeping through a given territory. Hence, the z-axis represents time. On these electronically reconstructed time mode™ scans, displacements of the myometrial-endometrial interphase take the appearance of periodic waves, with each wave individually representing an actual contraction of the uterus.

Follicular Phase During the follicular phase, there is a progressive increase in uterine contraction (UC) frequency under the influence of rising E2 levels,83,234,235 which culminates at approximately 5 UC per minute just prior to ovulation.73 Characteristically, only the subendometrial layers of the myometrium are involved in these contractile events, which are never perceived by women despite UC frequency being higher at that time than at any other time in the menstrual cycle.24 The majority of investigators,83,236 but not all,237 have reported a clear predominance of UC dysplaying retrograde cervix-to-fundus displacement during the late follicular phase. In these studies, UC displacement was evaluated on fast plays of ultrasound sequences, or by analyzing the propagation of the increase in the IUP signal.231 Yet, determining the UC direction is complicated by two unknowns: (1) While the contractile structure that is responsible for UCs is the subendometrial portion of the myometrium, the visualized phenomenon on ultrasound images is the result of the displacement of the endometrium. Hence, the parameter that is analyzed is an indirect reflection of the contractile process. (2) Ultrasound identifies the apparent retrograde displacement of contractile waves without establishing whether this does or does not result in true displacement of the uterine contents. In the case of sperm for example, retrograde transport will also depend on whether an appropriate opening takes place or does not take place at the utero-tubal junction when the contractile event reaches the junction. Retrograde UCs identified during the late follicular phase are thought to be instrumental in the rapid transport of sperm that takes place from the vagina to the pelvic cavity following intercourse in the late follicular phase.231,238,239 Thus, UCs and possible disruption of their patterns, may, therefore, play a key role in the reproductive process and in certain forms of infertility. However, as stated above,

echographic analyses of UC direction have never been shown to correlate with the actual displacement of uterine contents. Using 99Te labeled macro-albumin aggregates (MAA), Leyendecker’s team showed that contractions with retrograde displacement of uterine contents predominate during the late follicular phase.235 These authors also observed that transport takes place toward the tube facing the developing follicle.24,235 In various intervention trials, Leyendecker’s team reported evidence for the involvement of oxytocin (OT) in the retrograde contractions encountered during the late follicular phase.24,231 That OT, and possibly vasopressin (AVP), play a physiological role in late follicular phase UCs, and are instrumental in the control of sperm transport, remains to be determined. Retrograde displacement of uterine contents triggered by late follicular phase UCs appears to be disrupted in women suffering from endometriosis235 or intramural uterine fibroids.156,157 This could be responsible for the infertility that accompanies both conditions.

Luteal Phase During the luteal phase, the UC pattern is characterized by a state of uterine quiescence brought about by the smooth muscle relaxing properties of progesterone. In a prospective trial, Wilcox and colleagues240 showed that pregnancies never occurred when the single unprotected intercourse took place after the day of ovulation. This finding may be explained in part by the utero-relaxing properties of progesterone and the resulting decrease in uterine contractility, particularly the retrograde UCs that are thought to be involved in sperm transport. In experimental trials, exogenous E2 and progesterone reproduce the UC patterns encountered in the follicular and luteal phases, respectively (Fig. 35.19). Ayoubi and colleagues observed that physiological E2 replacement administered to young women suffering from premature ovarian failure (POF) resulted in UC

Uterine contraction (min)

CHAPTER 35  Pelvic Imaging in Reproductive Endocrinology

Progesterone dose 45 mg 90 mg 180 mg

6 5.5 5 4.5 4 3.5 3 2.5 2 1.5 1 15

16

A

17

18

20

Cycle day 5.5

Uterine contraction (min)

19

3D-derived method Fast play

5 4.5 4 3.5 3

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women at the time of menses. Moreover, UCs are commonly perceived by women at that stage of the cycle. At times, UCs occuring during the inter-cycle interval are painful enough to cause dysmenorrhea, characterized by painful uterine cramping. UC amplitude and resting tone are markedly higher during menses than at any other time during the menstrual cycle.230,233,241 These are the two parameters that are most increased in dysmenorrhea.241 Using MRI-based analyses, Kataoka and colleagues showed an enlargement of the subendometrial layer of the myometrium in dysmenorrheic women on cycle day 1, at a time when these women experienced strong cramping. Uterine images returned to normal on day 3 of the cycle when cramping had subsided (Fig. 35.20).232 In a prospective trial, Kido and colleagues compared the characteristics of MRI of the endometrium and subendometrial layer of the myometrium in 25 women on OC pills and 23 others having regular menstrual cycles.157 This trial revealed that endometrial distortion was significantly less prominent, and the subendometrial low intensity area significantly thinner, in the OC group who also presented a lesser degree of cramping as compared to their cycling counterparts.157 MRI is, therefore, the only imaging method providing noninvasive information on UCs that parallel clinical symptoms in dysmenorrhea.

Effects of Uterine Contractility on ART Outcome

2.5 2 15

B

16

17

18

19

20

Cycle day

FIGURE 35.19  Effects of exogenous progesterone on uterine contractility. A, In estrogenized women, exogenous administration of 45, 90, and 180 mg of progesterone/day resulted in a prompt decrease in uterine contraction (UC) frequency without dose-related differences. B, Direct measurements from fast plays of ultrasound sequences or using the 3D-derived method gave similar results. (Ayoubi JM, Fanchin R, Kaddouz D, et al. Uterorelaxing effects of vaginal progesterone: comparison of two methodologies for assessing uterine contraction frequency on ultrasound scans. Fertil Steril 76:736, 2001.)

frequency patterns that were similar to those encountered in the menstrual cycle particularly, during the follicular-luteal transition.83 Transvaginal administration of 45, 90, and 180 mg of progesterone per day using a sustained release vaginal gel, Crinone, resulted in a prompt decrease in UC frequency with no dosage-related differences. This indicates that following physiological estrogen exposure, minimal amounts of progesterone suffice to induce full utero-quiescence, as seen in the luteal phase of the menstrual cycle.83

Intercycle Interval During the intercycle interval (menses), uterine contractility increases in response to withdrawal from the uterorelaxing properties of progesterone upon demise of the corpus luteum, or discontinuation of exogenous progesterone or progestin administration. The role of concomitant withdrawal from E2 also produced by the corpus luteum and supplied in most OC preparations remains a matter for debate. In contrast to the late follicular phase, all layers of the myometrium are involved in UC experienced by

Ayoubi and colleagues83 were first to report an inverse correlation between UC frequency at the time of embryo transfer (ET) and IVF outcome (Fig. 35.21), a finding supported by data from others,242 but not all groups.243 More frequent uterine contractions had negative consequences for implantation and pregnancy rates. As UCs were measured prior to conducting embryo transfers (ETs), the study reflected the state of contractility prevailing prior to ET, not the impact of the ET procedure itself on contractility. In further trials, an inverse correlation between the progesterone levels on the day of ET and UC frequency was observed.116 Furthermore, an earlier onset of progesterone treatment on the day of oocyte retrieval lowered UC frequency on the day of ET, with a trend toward better pregnancy rates.83 Similarly, delaying ET to the fifth day after oocyte retrieval resulted in a profound reduction in UC frequency at the time of ET.83 Following extensive analyses of UC patterns as observed by ultrasound, Ijland and colleagues237 reported that women in whom UCs were predomanantly retrograde had higher pregnancy rates. The observation that certain IVF patients have increased UC frequency at the time of ET raises questions about the mechanism(s) at play. In a prospective trial, we studied UC frequency in the same patients during the menstrual cycle that preceded IVF and the IVF cycle itself.73 In these women, UC frequency reached a similar maximum on the day of the LH surge and hCG administration, despite E2 levels being markedly higher in the IVF cycle as compared to the menstrual cycle. This suggests that the E2 levels of the menstrual cycle already exert a maximal effect on UC frequency prior to the LH surge or hCG administration.73 As illustrated in Figure 35.22, a marked difference in UC frequency exists after ovulation. In the menstrual cycle, uterine quiescence induced by progesterone is observed as early as day 4 after the LH surge, with a mean UC frequency of 1.1 per minute. In contrast, UC

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A

B

FIGURE 35.20  Using magnetic resonance imaging (MRI), differences were observed in the junctional zone between women experiencing severe dysmenorrhea on day 1 of the menstrual cycle (A) and the same women assessed on day 3 when cramping had subsided (B). On day 1 a characteristic distortion of the endometrium (arrows) and a thickened junctional zone (arrowheads) was noticed, a finding that paralleled the intensity of pain perceived. On day 3, when pain had subsided, the endometrium and junctional zone had returned to normal. (From Kataoka M, Togashi K, Kido A, et al. Dysmenorrhea: evaluation with cine-mode-display MR imaging—initial experience. Radiology 235:124, 2005.)

Number

Pregnancy rate (%)

60

40

20

Menstrual cycle IVF

7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00

*Different from IVF **Different from baseline

* ** LH/hCG

0 ≤3

3.1-4

4.1-5

>5

UC/min FIGURE 35.21  Uterine contractility (UC) just prior to embryo transfer and pregnancy rates. In women who all had three good embryos available for transfer, an inverse correlation was observed between UC frequency and pregnancy rates. (From Fanchin R, Righini C, Olivennes F, et al. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod 13:1968, 1998.)

frequency remained high at 3.6 per minute on the 4th day after hCG administration, ultimately declining on day 6. Of note, however, these women started receiving exogenous progesterone on day 4 after hCG (after the UC measurement). Exogenous progesterone, therefore, may have been instrumental in the decrease in UC frequency observed on the 6th day after hCG in IVF. Taken together, these findings suggest that some degree of resistance to the uterorelaxing properties of progesterone exists in IVF, possibly related to high E2 levels. Pierzynski and colleagues228 provided pilot data suggesting that exogenous administration of the OT antagonist, atosiban, resulted in decreased UC frequency prior to ET. These findings open up new avenues for uterorelaxing therapy in relation to ET using OT and vasopressin receptor antagonists,244 when other uterorelaxants have failed.245

LH+2/retrieval LH+4/ET

** ** LH+6/ET+2

FIGURE 35.22  UC frequency was measured in the same patients, in the menstrual and IVF cycles. UC frequency was similar on the day of LH surge and hCG administration, indicating that the supraphysiological levels of E2 encountered in ART do not further stimulate UC frequency over what is encountered in the menstrual cycle. Following ovulation, UC frequency decreased earlier in the menstrual cycle than it did in ART, indicating that a relative degree of resistance to the uterorelaxing properties of progesterone exists in ART, most likely as a result of elevated E2 levels. UC, uterine contractility; E2, estradiol; ART, artificial reproduction technology; hCG, human chorionic gonadotropin; IVF, in vitro fertilization; LH, luteinizing hormone; ET, embryo transfer. (From Ayoubi JM, Fanchin R, Kaddouz D, et al. Uterorelaxing effects of vaginal progesterone: comparison of two methodologies for assessing uterine contraction frequency on ultrasound scans. Fertil Steril 76:736, 2001.)

Imaging Markers of Endometrial Receptivity The endometrial parameters assessed by uterine imaging that have been proposed as markers of endometrial receptivity are endometrial thickness and volume, echogenicity, Doppler perfusion parameters, and contractile patterns. Endometrial thickness reflects endometrial priming by estrogen necessary for an appropriate response to progesterone and, in turn, endometrial receptivity. Rashidi and colleagues246 reported no correlation between endometrial thickness on the day of hCG administration and pregnancy rate. Endometrial thickness was not different at 10.1 and 10.2 in pregnant and nonpregnant patients, respectively.

CHAPTER 35  Pelvic Imaging in Reproductive Endocrinology

Okohue and colleagues reported that an endometrial thickness 7 to 14 mm was associated with better outcome than when the endometrium was either less than 7 mm (P = 0.004) or greater than 14 mm (P < 0.0001),247 data in agreement with those of others.246 The early report by Casper’s group of poorer outcome in case of excessively thick endometrium100 was not confirmed in later studies, such as that of Richter and colleagues on 1294 patients.89 The possibility of positive bias towards a thicker endometrium could result from its relationship to heftier responses to COH, a parameter that is associated with better IVF outcome.248 By extension, a positive bias for Doppler findings of higher perfusion could be the consequence of an association with a more robust ovarian responses to hMG and FSH, and consequently increased oocyte quantity and quality. Whether different ovarian stimulation protocols had different impacts on endometrial thickness was loooked at by Imoedemhe and colleagues.249 These authors compared the effects of regimens using clomiphene citrate (CC) alone (regimen I) or in combination with 75 IU (regimen II), or 150 IU of hMG (regimen III), and found no differences in endometrial thickness. Raga and colleagues examined the predictive value of 3D endometrial volume calculations for the establishment of pregnancy in ART.11 In a study on 72 IVF cycles, pregnancy rates were significantly lower when endometrial volume was less than 2 mL as compared to 2 to 4 mL or greater than 4 mL. Zohav and colleagues found no differences in endometrial volume in 60 women pregnant through IVF, but observed that endometrial volume of less than 2 mL was associated with a significant increase in pregnancy loss.248 A recent study on E2 and progesterone cycles for frozen embryo transfers came to similar conclusions that lower volumes are an ominous sign.75 Ferriani’s group reported on endometrial volume measured one week after ET.250 A significant difference was found between the endometrial volume of pregnant (6.49 ± 1.97 mL) and nonpregnant women (3.4 ± 1.1 mL) and, but to a lesser extent, endometrial thickness. This latter observation of a difference in endometrial volume by a factor of nearly 2, 1 week after ET, between pregnant and nonpregnant women likely reflects a direct early influence of the developing pregnancy on the endometrium rather than preexisting differences. This finding is in agreement with the report of an early increase in E2 production that even precedes that of hCG in the late luteal phase of conception cycles.251 Endometrial echogenicity or ‘pattern’ has been proposed as a marker of endometrial receptivity.252 In early work, Casper’s team, who observed no differences in endometrial thickness between pregnant and nonpregnant women, reported three different patterns of endometrial echogenicity on the day after hCG administration.107 In these investigators’ hands, a hypoechoic endometrium carried a good prognosis. Numerous publications support the notion that a hypoechoic endometrium with a full three-line pattern is associated with good ART outcomes,108-113 although some failed to find a good predictive value of endometrial echogenicity.114,115 According to Rosenwaks’ team, a hyperechoic endometrium is a particularly ominous sign in women exposed to DES.253 Pregnancy outcome relative to endometrial thickness and pattern was evaluated in 540 cycles of IVF including DES (n = 50) and non-DES-exposed (n = 490) women.

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Endometrial patterns were designated as p1 = solid; p2 = ring; and p3 = intermediate. DES patients exhibited the ‘p1’ pattern more often than the majority of the non-DES-exposed group. There was no significant difference in endometrial thickness among the cycles where ‘p1’ was noted when comparing the DES (10.3 mm) with the non-DES-exposed (10.7 mm) groups. Notably, within the group exhibiting ‘p1’, no pregnancies occurred in the 18 cycles of DES-exposed women compared with a 39.2% clinical pregnancy and 36.5% delivery rate in the non-DESexposed controls (P <0.0001 and P = 0.008, respectively). The impact of uterine shape on pregnancy outcome was also investigated. A T-shaped uterine configuration was noted in 11 out of 18 (61.1%) cycles of DES-exposed women with pattern ‘p1’ compared with 9 out of 23 (39.1%) with pattern ‘p2’. Of cycles where a T-shaped uterus was demonstrated, none out of 11 (0%) with pattern ‘p1’ compared with 4 out of 9 (44.4%) with pattern ‘p2’ resulted in pregnancy (P = 0.026). These data suggest that the endometrial pattern is one of the most significant variables for pregnancy outcome in DES-exposed women undergoing IVF. The two last parameters proposed as markers of endometrial receptivity are Doppler data on endometrial and subendometrial vascularization and uterine contractility. Early Doppler studies that claimed to identify flow parameters that precluded the possibility of embryo implantation were never verified. Instead, all available studies that used either pulsed Doppler data or the more sophisticated and operator independent 3D-PDA failed to find differences between conception and nonconception cycles.5 These findings showing that Doppler data—even using the newest and most sophisticated techniques—are not predictive of ART outcome were confirmed by recent reports.21,254 Hence, Doppler data cannot be considered to be a marker of endometrial receptivity. In our experience, increased uterine contractility portends poor embryo implantation rates.221 These findings have been confirmed by some researchers,228 but have been challenged by others.243

Embryo Transfers ET should be performed with utmost care. Sloppy practices and imprecise placement of the transferred embryos in the uterine cavity undermine the good clinical and biological work that came together to obtain these embryos.255 Interestingly, it is the very person who first conceived oocyte retrieval under ultrasound guidance256 who was the first to advocate in 1987 for ultrasound-directed embryo transfers.4 In 1991, a prospective trial reported higher pregnancy rates in 94 women who had an ultrasound-guided transfer as compared to 246 controls who had the classical approach. Similar benefit was found in donor egg recipients. In a retrospective comparison of 137 embryo transfers, Lindheim and colleagues showed that pregnancy and embryo implantation rates were higher in women whose transfers were ultrasound-guided as compared to their counterparts in whom ultrasound was not used,257 a finding confirmed by Coroleu and colleagues with even greater differences.258 These pioneering studies were later supported by numerous others, including sets of meta-analyses,259-262 although a few reports failed to document the superiority of ultrasound-guided transfers.263-265 Ata and Urman argued against the conclusions

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of non-superiority.266 They noted that no ultrasound-guided trial reported lower results than the classical “clinical touch” method. Ata and Urman also underscored the fact that not all ultrasound-guided trials placed the embryos where their chances of implantation were optimal.266 Flisser and Grifo, whose own study showed no improvement in pregnancy rates with ultrasound-guided transfers, nonetheless recommended systematic use of ultrasound,263 since it is impossible to preemptively determine who is likely to benefit from ultrasoundguided ET. A recent meta-analysis that retained 17 trials out of 59 identifed in the literature on ultrasound-guided transfers. In 7 out of 17 of these trials that reported on clinical pregnancy rate, ultrasound-guided ET resulted in improved ART outcomes.267 Bodri and colleagues suggested that vaginal ultrasound may be used as effectively as the abdominal approach, while providing further comfort for the patient.268 With the ability to visualize the tip of the ET catheter, ultrasound-guided transfers beg the question: Which is the optimal location in the uterine cavity for transfer? Data from prospective trials unveiled a counterintuitive scenario: ETs are more successful when performed low rather than high in the uterine cavity. As early as 2002, Coroleu and colleagues reported a prospective trial in which embryos were transferred in 180 consecutive IVF patients at 1, 1.5, and 2 cm from the fundus.259 Unequivocally, embryo transfers performed 2 mm and 1.5 mm from the fundus resulted in higher pregnancy rates than seen when embryos were placed 1 mm from the fundus. In agreement with these findings, Pope and colleagues observed in a retrospective analysis that the further away the tip of the catheter was from the fundus, the higher the pregnancy rate.269 A regression analysis in this trial indicated that each millimeter in excess of 1 cm from the fundus translated in an 11% increase in pregnancy rate, a trend later confirmed by others.5 As interest in ultrasound-guided transfers gained momentum, industry designed catheters specially equipped with echogenic tips that facilitate visibility on ultrasound. A randomized controlled trial (RCT) comparing a standard (n = 95) and a new echogenic soft Wallace catheter (n = 98) revealed a trend (P = 0.08) for higher pregnancy rates with the echogenic catheter,270 a finding confirmed by others,271 but not all studies.21 It appears, therefore, that while new echogenic catheters are easier to visualize, they do not necessarily bring higher pregnancy rates. Fang and colleagues recommended verifyng the proper positioning of the catheter on 3D-­ultrasounds for fear that if the tip deviated from the 2D-plane it might not be properly identified.272 These authors thoughtfully claim that in such a situation, the tip might be actually more advanced, which would be detriemental. The same argument would favor a catheter having its echogenic mark limited to its tip rather than full length of the catheter for best assurance that the echogenic mark represents the true tip of the catheter. A closing comment regarding the competitive comparisons between various ET catheters is offered by Yao and colleagues, who report that the results of such comparisons are operator-dependent.273 In 1446 embryo transfers performed in 1155 women undergoing ART, 723 cycles were randomized to ‘soft’ (Cook) and 723 others to ‘semisoft’ catheters (Frydman®). Overall, the odds ratio of clinical pregnancy for the Cook versus the Frydman catheter were not different at 1.11 (95% CI: 0.89 to 1.38). Yet, these differed significantly for two of the three operators, being at 1.19 (95%

CI 0.84 t 1.69), 2.35 (95% CI 1.40 to 3.95), and 0.69 (95% CI 0.48 to 0.99) for each of them.273 Using ultrasound-guided ET in routine ART practice is a valuable tool for teaching the procedure to new operators.274 At our institution, new operators have to demonstrate competance at performing mock ETs under ultrasound guidance before moving on to actually performing true ETs.

Ovaries Unlike the uterus, which reaches a definitive and stable size at the time of puberty (short of developing tumors like fibroids), the ovaries change in size and aspect throughout the different phases of their function. Transvaginal ultrasound provides an unrestricted view of the ovaries with the fluid-filled follicles presenting a characteristic echographic signature to this organ. This makes ultrasound the first choice imaging approach for exploring the ovaries, even in cases of obesity. Ultrasound identifies two ovarian constituents: the cortex and stroma.165,275,276 Following a description by Pache and colleagues,277 the total count of 2 to 10 mm follicles in each ovary constitutes the antral follicle count (AFC).278 Remarkably, the AFC shows little intracycle variation,279 and is not affected by hormonal treatments such as the OC pill.279,280 Granulosa cells of antral follicles are endowed with FSH receptors, rendering these the cohort of follicles capable of responding to rising FSH. They are, therefore, the recruitable follicles.281 Recruitment by FSH takes place either through the physiological intercycle FSH signal or with exogenous FSH and hMG treatments administered in the context of ART. Therefore, the number of antral follicle—the AFC—will affect the magnitude of the ovarian responses to stimulation in ART or COS.281,282 We know from the work of Gougeon’s team135,283 that the cohort of antral follicles identified on ultrasound examination represents a constant fraction of the pool of primordial follicles remaining in the ovary at any given time (Fig. 35.23).284 Thus, there are grounds for assuming that

10 mm

AFC FIGURE 35.23  The antral follicle count (AFC). While the AFC does not fluctuate during the menstrual cycle, computation is classically done at baseline of the menstrual cycle (cycle day 2 to 4), because measurements are not hindered by a growing follicle or a corpus luteum. Traditionally, all follicles of 2 to 10 mm are counted.

CHAPTER 35  Pelvic Imaging in Reproductive Endocrinology

the AFC reflects the number of remaining follicles, or ovarian reserve.168,285 Therefore, the AFC aims at predicting: (1) the amplitude of the ovarian response to exogenous FSH and hMG or COS (for singling out excessive and poor responses and, therefore, adjustment of the doses of FSH and hMG accordingly) and; (2) ART outcome, on the assumption that it depends on the number of oocytes retrieved.286 The latter point begs a question about whether oocyte quantity and quality are inherently linked or not. As discussed below, the nature of this link between oocyte quantity and quality differs based on whether a reduction in the AFC is age-dependent or independent. The echographic characteristics of the ovaries (volume and/or AFC) are now taken into account when making the diagnosis of PCOS, per the joint ESHRE/ASRM Rotterdam consensus conference.82 Ovarian volume and/or AFC represent one of the two of three criteria necessary for making the diagnosis of PCOS. Large changes in blood flow take place in the ovaries during physiological (from follicular maturation to corpus luteum formation)132 and abnormal function (ovarian stromal flow in PCOS),287,288 or in the context of specific pathologies such as cancer.289,290

Ovarian Anatomy and Functional Changes The Ovary Before and After Puberty Ovarian growth in infants and children reflects functional changes rather than chronological age. In a prospective trial, Buzi and colleagues looked at 117 normal girls between the ages of 1.1 and 15.6 years, and 87 girls presenting with premature sexual maturation.291 In girls showing no signs of abnormal sexual maturation, ovarian volume was 1.1, 2.3, and 5.3 mL in prepubertal, pubertal, and postmenarchial girls, respectively. These changes correlated better with Tanner stages of sexual development than with chronological age.292 In infancy and childhood, follicles begin their maturation and rapidly become atretic in the absence of gonadotropin stimulation, giving the ovary a microcystic pattern.34,293 Before secondary sexual characteristics become visible, puberty-related changes take place with the ovaries showing changes in their morphology characterized by the presence Early reproductive life

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of several follicles of 4 to 9 mm in size.34 This appearance, sometimes referred to as megalocystic or multicystic,294,295 needs to be distinguished from the polycystic ovary.296-298

The Ovary in the Menstrual Cycle The intercycle interval constitutes the baseline state of the ovary in the menstrual cycle. Therefore, ovarian scans have been timed using this reference point for measuring the AFC for assessing the ovarian reserve and for predicting the response to COS in ART (Fig. 35.24). During the intercycle interval, the small 2 to 9 mm follicles, computed in the AFC, are the follicles capable of responding to FSH, whether naturally occurring or part of COS in ART. Through daily evaluation of individual ovarian follicles on repeated transvaginal scans, Pierson’s team identified bursts of synchronous growth in a cohort of follicles, or waves of follicle development.299 These investigators identified minor waves of follicular growth leading to all the follicles ultimately undergoing atresia and major ones leading to further follicular growth and single follicular dominance.299 The nature of the mechanisms controlling these successive waves encountered during the menstrual cycle still evades our understanding. Follicular growth leading to follicular dominance can be ascertained by viewing follicles greater than 10 mm. From repeated measurements, it was determined that follicles reach the 12 to 13 mm mark on average by day 9 of the menstrual cycle.299 At this stage, granulosa cells acquire LH receptors with evidence that follicular maturation can be promoted with LH only300 or mini doses of hCG.301-304 Follicular growth rate appears fairly constant in the menstrual cycle once follicles are greater than 13 mm, with a daily mean increment in diameter of 1.4 to 1.5 mm/day. Follicular growth is accelerated in COH, however, where it reaches 1.6 to 1.8 mm/day.305 The side of ovulation in the menstrual cycle and whether this regularly alternates from one cycle to the next has been a matter of enduring debate. Some reports based on histological evidence support the concept of alternating sides,306 while others claim that it is not more frequent than expected by chance alone.307 Finally, some propose that the side of follicular growth and ovulation only alternates in cases of short cycles,308 while the ovulation side is randomly

Late reproductive life

Antral follicles

FIGURE 35.24  Nongrowing and antral follicles in early and late reproductive life. In early and late reproductive life, the antral follicle count (AFC) reflects the total number of follicles remaining, or ovarian reserve. (From Hansen KR, Knowlton NS, Thyer AC, et al. A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause. Hum Reprod 23:699, 2008.)

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determined in cases of long cycles. Reassessing this latter paradigm, Fukuda and colleagues studied 410 natural cycles of 123 infertile women undergoing 267 IUI and 143 IVF natural-cycle treatments, respectively.309 In this population, ovulation occurred contralaterally by reference to the preceding cycle in 57% of the 410 cycles, but in 72% of women whose follicular phase was less than 13 days. Conversely, ovulation occurred randomly on either side when the follicular phase was greater than 14 days, thereby giving credence to the concept that the side of ovulation only alternates in short cycles. From these authors’ observation, ovulation appeared of better quality when occurring on the contralateral side with reference to the previous ovulation with better outcomes in both IUI and IVF cycles. This supports the concept that ovulation is of better “quality” when the dominant follicle is contralateral to the prior ovulation as compared to ipsilateral. Moreover, significantly higher estradiol to androstenedione and estradiol to testosterone ratios and lower androstenedione levels were observed in contralateral as compared to ipsilateral ovulation cycles. Practically speaking, therefore, the chance of conceiving during a natural cycle may be affected by the site of ovulation in the preceding cycle. The side of ovulation, however, did not influence Doppler blood flow in the ovarian stroma or follicular and uterine arteries.310 Pierson’s team prospectively assessed imaging characteristics of the corpus luteum (CL) with daily ultrasounds in 50 normo-ovulatory women.311 The CL was detectable on ultrasound scans in 100% of women during the luteal phase, with persistent structures found in 90% of women during the subsequent follicular phase. Two days after ovulation, a central fluid filled cavity was present in 88% of the cases, declining to 34% after 13 days and 2% after 27 days.307

The Ovary during Hormonal Contraception The impact of OC treatment on ovarian follicles depends on the size of the follicles when OC treatment is initiated. In a prospective trial, Baerwald and colleagues determined that no follicular growth took place when all the follicles were less than 10 mm at the time of starting OC pills.312 Conversely, approximately 30% of follicles that were greater than 14 mm at the onset of OC treatment went on to ovulate, while the rest regressed and became atretic. In parallel work, these investigators determined that follicular development takes place during the 1-week hormone-free interval of classical OC pill regimens.313 In a trial looking at normal volunteers on OC pills, they determined that nearly half (47%) of women grew follicles greater than 10 mm, with 53% of them having follicles reaching 14 mm in diameter and an increase in E2 production. Contrasting with these findings, echographic assessments showed similar rates of follicular growth in women using progestin only or barrier contraception in the postpartum period. Endometrial thickness was markedly reduced, however, in the progestin only contraception group, indicating that this form of contraception acts on the uterus and cervix rather than by blocking ovulation.314 Hormonal contraception causes a significant reduction in ovarian volume in normally cycling women315 and in women suffering from PCOS.290,316 In cycling women, the effect of hormonal contraception on ovarian volume was more profound in cases of continuous rather than cyclical administration of hormonal contraception.317 Remarkably, however,

AFC and AMH values remained unaltered in PCOS women receiving hormonal contraception, in spite of the reduction in ovarian volume observed.290

The Ovary Through Functional Aging and Menopausal Changes Ovarian volume decreases with age. Early on in the highresolution ultrasound era, their was optimism about the possible predictive value of ovarian volume measurements for future reproductive performance. Surveys of published data indicated that ovarian volume was a good and easily obtainable index of the primordial follicle population based on comparisons with Faddy’s data.318 Pavlik and colleagues retrospectively reviewed data from 58,673 ovarian scans obtained from 13,963 women 25 to 91 years of age who underwent annual scanning as part of ovarian cancer screening program. Ovarian volume was calculated using the prolate ellipsoid formula (L × H × W × 0.523).123 Mean ovarian volume was 6.6 ± 0.19 cm3 in women younger than 30 years old with a decrease that reached statistical significance in women between the ages of 30 and 39 years old. Yet, the practicality of ovarian volume measurements for predicting ovarian reserve on an individual basis is somewhat limited, as clinically meaningful changes only materialize when women are at the boundaries of their entrance into the naturally infertile period of life. In Pavlik’s analysis, it is in the group of women between 40 and 49 years of age that ovarian volume was found to be markedly reduced at 4.8 ± 0.3cm3. Ovarian volume decreased to 2.6 ± 0.01 cm3 in postmenopausal women 50 to 59 years of age, with further decreases observed during the sixth and seventh decades of life.123 In their extensive study, these authors also looked at relationships between height and ovarian volume. Their findings with no progressive increase with height, but a statistically significant break with higher volumes in women over 5 feet 8 inches tall. There was also a statistically significant reduction in ovarian volume in women younger than 50 years old taking estrogens, but not in postmenopausal women. In a retrospective chart review, Syrop and colleagues looked at possible different predictive values of the volume of the smallest and largest ovaries in individuals.319 Their results indicated that the volume of the smallest ovary was a better predictor of peak E2 levels and numbers of oocytes and embryos and clinical pregnancy in IVF. By current definition, premature ovarian failure (POF) occurs when ovarian functions fail before the age of 40. This implies a complete form of hypergonadotropic hypogonadism with an implicit loss of fecundity. More commonly encountered in everyday ART practice are cases of premature ovarian insufficiency or occult POF (oPOF), which hide behind seemingly normal ovulatory cycles. In oPOF, the menstrual cycle is maintained and seemingly ovulatory, with no anomaly except maybe for a shortening of the cycle duration with a characteristically early ovulation. Embedded amongst the classical POF and oPOF that results from early ovarian aging, there are sporadic cases of a variant disorder characterized by insensitive ovaries. The difference between oPOF and POF on the one hand, and the rare cases of insensitive ovaries on the other, is more than just an issue of terminology, as pregnancy is much more likely to be achieved in the case of insensitive ovaries. Assessing ovarian volume in POF or oPOF may identify the

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subgroup of women suffering from insensitive ovaries for whom therapeutic efforts can be undertaken. In a prospective trial, Mehta and colleagues looked at 17 women with fully expressed POF.320 Based on the ecographic appearance of their ovaries, these authors sorted POF women into two groups, with (n = 7) and without ovarian follicles (n =10). Hormonal parameters (FSH and E2) and endometrial thickness were identical in the two groups, whereas ovarian volume was markedly larger at 2.8 ± 0.4 mL than in the no ovarian follicle group (1.4 ± 0.2 mL). On average, two follicles were identified in the “with follicles” group, whereas by definition none were identified in the “no follicle” group. These authors contend that ultrasound imaging of the ovaries allows identification of a subgroup of women presenting with hypergonadotropic hypogonadism who suffer from insensitive ovaries, rather than POF. Pregnancies are more likely to occur in insensitive ovaries than they are in POF following treatments aimed at lowering endgenous gonadotropins in order to resensitize FSH receptors. This is can be achieved by 2- to 3-month treatment with E2 and progesterone for lowering FSH levels followed by FSH administration. Falsetti and colleagues observed normal ovarian volumes (3.1 ± 0.3 mL) in 14 out of 40 (35%) women with frank POF, whereas ovarian volume was significantly smaller in the remaining 26 (65%) women.321 In all forms of POF and more importantly oPOF, screening for possible premutations of the FMR1 gene should be considered. Ignored, these may transform to the full mutation in the next generation, possibly causing Fragile X syndrome in male offspring.322 In Turner syndrome, the number of primordial follicles has been estimated to be normal until at least the 18th week of fetal life, with uncertainties as to the time when atresia starts to accelerate.323 The possibility of transplanting cryopreserved ovaries,324 including the report of livebirths,325 has sparked interest in cryopreserving ovaries from Turner patients before follicle depletion is completed.

In a prospective trial, Hreinsson and colleagues reported ovarian follicles present in eight of nine adolescent girls diagnosed with Turner syndrome, but who all underwent spontaneous onset of puberty.326 In these adolescent girls, the density of primordial follicles in ovarian samples studied surgically showed an inverse correlation with plasma FSH levels. It would be probably more useful to cryopreserve ovaries in infancy or childhood, when the pool of primordial follicles remaining is likely to be much larger, provided that the diagnosis of Turner syndrome can be made that early. Unfortunately, normative data for infant and child ovary imaging (transvesical) have not been established, which creates challenges in the assessment of subjects who might be candidates for ovarian cryopreservation. Assessing ovarian volume and particularly, differences in size between the two ovaries is useful when screening for ovarian cancer amongst other echographic criteria.327 Based on their echographic analyses of postmenopausal ovaries, Campbell and colleagues concluded that the percentage mean difference between the two ovaries was 42.88% ± 32.05%.328 These authors concluded that any finding showing an ovary with a volume that is greater than twice the size of the other ovary should be considered as suspicious of ovarian cancer.

Antral Follicle Count: A View of the Ovarian Reserve For each woman, the endowment of primary oocytes is thought to be finite and established during intrauterine life. It progressively decreases to reach approximately 2 million at birth and 400,000 at puberty. Drawing from extensive histological studies of ovaries from women of all ages, Faddy and colleagues described a progressive decay of primordial follicles over time that followed a bimodal pattern (Fig. 35.25).283 They identified a slow depletion rate that prevails until the age of approximately 37 years old. Then, 8

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FIGURE 35.25  A, Depletion of primordial follicles over time. In their analysis, Faddy and colleagues observed an exponential decline of primordial follicles in a biphasic pattern. The first exponential rate parameter was calculated at –0.097 and the second at –0.237. The change from the first (slow decay) to the second (rapid decay) rate of follicular decline occurs when the number of remaining follicles falls under the critical figure of 25,000. On average this transition from the slow to the rapid follicular decline occurs at age 37.5 years, but occurs earlier in cases of of surgery for ovarian cyst. B, In contrast to the bimodal mode of follicular depletion, Hansen and colleagues propose a model with no sudden change in the rate of follicular decay, but rather a constantly increasing pattern. (A, From Faddy MJ, Gosden RG, Gougeon A, et al. Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod 7:1342, 1992. B, From Hansen KR, Knowlton NS, Thyer AC, et al. A new model of reproductive aging: the decline in ovarian nongrowing follicle number from birth to menopause. Hum Reprod 23:699, 2008.)

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FIGURE 35.26  Depletion of antral follicles over time. Regression lines of inhibin B (log scale), antral follicular count (AFC), and anti-müllerian hormone (AMH) (log scale) values by age for subgroups of young hypergonadotropic women. °-°-°, Controls; +-+-+, incipient ovarian failure (IOF); transient ovarian failure (TOF); Δ-Δ-Δ, premature ovarian failure (POF). (From Knauff EA, Eijkemans MJ, Lambalk CB, et al. Anti-müllerian hormone, inhibin B, and antral follicle count in young women with ovarian failure. J Clin Endocrinol Metab 94[3]:786–792, 2009 Mar.)

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FIGURE 35.27  Method of AFC measurement. The AFC can be computed from 2D or 3D images. In the latter case, antral follicles can be counted using the multiplanar approach, scanning through the ovarian volume, or by automated analysis. The latter relies on an automatic antral follicle recognition system using the Sono-Automatic Volume Count technique based on a 3D-inversion mode rendering method. Using software that individually color codes each identified follicle and provides an objective measurement of its mean and absolute diameters and its volume. A, The initial fully automated assessment missed several antral follicles. B, The same ovary after these missed follicles have been manually identified through postprocessing. (From Deb S, Jayaprakasan K, Campbell BK, et al. Intraobserver and interobserver reliability of automated antral follicle counts made using three-dimensional ultrasound and SonoAVC. Ultrasound Obstet Gynecol 33[4]:477–483, 2009 Apr.)

when the number of oocytes remaining becomes less than 25,000, an abrupt increase in oocyte depletion rate appears to take place, or fast depletion rate. The change from the slow to the fast depletion rate of primordial follicles takes place at approximately the age of 37 years. These authors suggested, however, that it is the number of remaining follicles—the critical number being 25,000—that triggers the acceleration in the depletion rate, not the age, per se. However, the claim that follicular depletion over time follows a bimodal pattern has been challenged by Hansen and colleagues.284 These latter authors contend, on mathematical grounds, that follicular depletion over time follows a progressive acceleration pattern described by a power function, rather than the bimodal model (Fig. 35.26). The methodology for AFC measurement has been the source debate. Both 2D or 3D approaches for AFC measurements showed good overall interobserver and intraobserver

reliability.329 Recently, a novel method of AFC assessment was developed based on automatic isolaton volumes of low echogenicity—the antral follicles—known as the 3D inversion mode rendering or SONO-AVC® method330 (Fig. 35.27). AFC measurements made by classical 2D- or 3D-based approaches revealed that automated measurements took slightly more time and in general yielded lower values.331 Jayaprakasan and colleagues determined that the variability AFC was significantly lower with inversion mode, while it was equivalent when measured with the 3D multiplanar and 2D equivalent methods. Yet, the authors determined that interobserver reliability was affected by image quality when using the 3D-rendered inversion method.332 Moreover, mean time for measurement varied greatly with the methods used, ranging from 34 to 50 to 216 seconds for the three methods.332 AFC has been found to correlate with the number of oocytes retrieved in COH333-335 and risk of developing

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OHSS.336 In a prospective trial, AFC was found superior to basal FSH for predicting ovarian response to COH.337 Ng and colleagues336a compared AFC to serum FSH measured at baseline and post-clomiphene citrate challenge test (CCCT) for their predictive values regarding ART outcome. These authors found that AFC that remained unchanged after stimulation (CCCT) or after GnRH down-regulation, had the best predictive value for the quantitative outcome of COH expressed by the number of oocytes retrieved. This was followed by combined baseline and post-CCCT FSH concentration and age of the woman. Baseline and post-CCCT FSH concentration was a slightly better predictor of E2 values. In a prospective trial, Jayaprakasan and colleagues looked at the predictive value of AFC assessed from 3D data and compared their data to baseline FSH levels. One of three methods of measurement was used, including the new 3D-rendered inversion mode.332 These authors’ results indicated that total AFC was the best predictor of the number of oocytes retrieved in ART followed by baseline FSH. Moreover, AFC was significantly lower in the nonpregnant group. AFC less than 7 (less than 6 using 3D-rendered inversion mode) predicted poor ovarian response leading to cycle cancellation with sensitivity of 100% and specificity ranging from 93% to 96%, depending on the method used. Prediction of nonconception was low for all methods of AFC measurement. Data obtained from 3D-rendered inversion mode were the closest to the actual number of oocytes retrieved. More recently, SONO—AVC has been used for an automated assessment of the growing follicles in ART, which was demonstrated to be reproducible and reliable,338 as well as acruate.338 Haadsma and colleagues proposed that amongst the cohort of 2 to 10 mm follicles retained for AFC computation, the subgroups of smaller and larger antral follicles had different predictive values.339 In a cohort of 474 women, the number of very small follicles (2 to 6 mm) declined with age, while that of larger follicles (7 to 10 mm) remained fairly constant over time. Independent of age, the number of small follicles was related to the results of ovarian reserve tests. These authors claimed that the number of small antral follicle represents the functional ovarian reserve. Along these lines, Scheffer and colleagues observed a steeper yearly decline in the number of small antral follicles (2 to 5 mm) as compared to that seen for larger follicles (6 to 10 mm).340 Because a direct correlation has been established between the size of the antral follicle cohort and the number of remaining primordial follicles,135 interest has been focused on determining the rate of depletion of antral follicles over time. In a prospective trial, Ng and colleagues determined that the rate of AFC decrease per year was of 0.35 follicle per year (95% CI 0.26, 0.45).340a These findings are of similar magnitude as the decline rate of 0.52 follicle per year (95% CI 0.26, 0.45) observed by the same group in a subsequent study conducted in a different cohort of women.82 Yet, both results were obtained in Chinese women, and other publications reported faster AFC decays in Caucasian women where an annual loss of 0.95 follicles was observed,341 or 8.2%.342 There are no current explanations for the lower AFC losses observed in Chinese women as compared to their Caucasian counterparts. Hendriks and colleagues conducted a meta-analysis looking at the respective values of ovarian volume versus AFC

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FIGURE 35.28  Prediction of ovarian response to COS and ART outcome. Ovarian reserve parameters (AFC calculation on ultrasound and AMH and FSH levels) predict the ovarian response to COS (data shown for poor response), but not ART outcome (data shown for non-pregnancy prediction). ROC curves of studies reporting on the performance of the AFC, AMH, basal FSH, and multivariate (MV) models to predict poor ovarian response during ovarian stimulation in IVF (A) and the occurrence of pregnancy (B). sROC, summary receiver operator characteristics; AFC, curves for antral follicle count; AMH, anti-müllerian hormone; FSH, follicle stimulating hormone; MV, mean volume of the ovaries. (From Broekmans FJ, Soules MR, Fauser BC. Ovarian aging: mechanisms and clinical consequences. Endocr Rev 30[5]:465–493, 2009 Aug.)

for predicting IVF outcome.343 A total of 17 studies, 11 from prior meta-analysis337 and 6 new ones were retained for looking at the predictive value of AFC. Comparison of ROC curves showed that the prediction of poor response was significantly better for AFC than for ovarian volume (P <0.005) (Fig. 35.28). The prediction of nonpregnancy was poor for both tests, but the performance of AFC was slightly better than that of ovarian volume. This could have been predicted from the fact that both tests represent the quantitative aspect of ovarian reserve.

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Sensitive assays for AMH, also referred to as müllerian inhibiting substance (MIS), have revealed that AMH is produced by growing pre-antral follicles.344 The cohort of AMH-producing follicles (100 to 200) is markedly larger than that of antral follicles (10-20). Yet, both reflect the pool of primordial follicles present at any given time. From Gougeon’s work,135,283 and that of others,332 the cohort of antral follicles is believed to remain constant throughout the menstrual cycle, thus providing constant AFC throughout the menstrual cycle and limited intercycle variability as compared to day-3 FSH levels. Likewise, AMH levels show a high degree of correlation with AFC,345 and remain essentially constant throughout the menstrual cycle346-348 with limited intercycle variability.73,349,350 AMH levels are not affected by prolonged treatment with oral contraceptives348 in spite of the profound decrease observed in ovarian volume,316 and remain constant in pregnancy and the postpartum period,351 and following FSH administration in regularly ovulating women352 and women suffering from PCOS.353 Possible post-ovulatory fluctuations in AMH levels have been suggested.354 We concluded that this discrepancy did not result from methodological differences, and that if a slight postovulation decrease truly occurs, it is of lesser magnitude than intercycle variability of AMH measurements and can, therefore, be ignored.322 Recent studies have questioned whether AMH measurements add any value over simple AFC determination.343 AMH does offer practical advantages for identifying PCOS amongst women whose AFC are so elevated that follicles are too numerous to count, or when AFC cannot be obtained. In conclusion, AFC is a readily available imaging-­ dependent index of ovarian reserve, which can predict ovarian responses to COS in ART. In the normal ovarian aging process, the decrease in the number of oocytes is paralleled by a decrease in oocyte quality. In this context, a correlation exists between lower AFC and lower oocyte quality. Yet, this link is not an inherent one, but rather reflects the confounding effects of age.

Ovarian Perfusion Perfusion of the Maturing Follicle and Corpus Luteum (CL) Power Doppler has revealed the development of vessels in the wall of the dominant follicles in the later stages of the follicular phase355 and in COH,356-358 and a possible link with oocyte quality and reproductive outcome. Characteristically, this vascular development gives a colored annular ring that circles the maturing follicle. Typically, this is observed around certain but not all follicles developing in COH, starting on average 1 to 2 days prior to the time when hCG is commonly administered in COH (Fig. 35.29).359 Claims have been made that oocytes originating from well-vascularized follicles yield better reproductive outcomes than their counterparts originating from less vascularized follicles.86,360,361 In line with this notion, a decrease in follicular blood flow in aging ovaries has been reported.362 However, these results were challenged by Ragni and colleagues.363 In a prospective trial on 318 women, these authors found that vascularity of the developing follicle did

not predict the chance of pregnancy in women undergoing mild COS and IUI cycles. Pregnancy rates in the low-, medium-, and high-grade vascularity groups were 14.1, 10.0, and 11.8%, respectively. Lozano and colleagues examined how follicular vascularization correlates with oocyte quality.364 In their study, 61 normally ovulating women were prospectively studied. Their treatment consisted of daily GnRH antagonist (Cetrotid 0.5 mg/day) and 150 IU of hMG, starting when a growing follicle greater than 12 mm was observed. Ovulation was triggered as soon as the single dominant follicle reached 16 mm in diameter. Follicular vascularization was assessed just prior to oocyte retrieval, 34 to 36 hours after hCG (5000 IU) administration. Using 3D-based methodology, the vascularization index (VI) and flow index (FI) were taken as quantitative and qualitative reflections, respectively, of follicular vascularization. VI bore no predictive value to IVF outcome, but FI strongly predicted positive clinical pregnancies. These authors concluded that a qualitative rather than quantitative relationship exists between follicular vascularization and oocyte quality. After ovulation the developing CL is the site of an intense neovascularization process, rapidly becomes the most vascularized organ of the body on a blood flow per tissue volume basis. On color and power Doppler images, the CL acquires the characteristic “ring of fire” appearance (Fig. 35.30). In a longitudinal study throughout the luteal phase and early pregnancy, Tamura and colleagues showed that the resistance index (luteal RI) was associated with corpus luteum function.365 Claims have been made that correlations exist between CL Doppler data and quality of the luteal phase,366 leading to the proposal that Doppler data could be used for assessing the luteal phase367 and predicting pregnancy outcome.368 In spontaneously occurring singleton pregnancies, Frates and colleagues failed to find a correlation between the echographic characteristics of the CL and first trimester pregnancy outcome.368 On gray-scale images, we believe that an educated eye can easily identify the fine dual contours of vessels that run around the CL, an impression which is supported by the finding of a correlation between Doppler data and vascular density.369 It should be noted that the increased vascularity of the CL may at times be confused with the vascular ring that characterizes certain ectopic pregnancies.370

Perfusion of the Ovarian Stroma Color Doppler has revealed the presence of ovarian stromal vascularization. From the outset, it was suspected that a difference in the degree of vascularization exists between normally ovulating and PCOS women. According to Battaglia and colleagues, ovarian stromal vascularization is identified in 50% and 88% of the cases in normally cycling and PCOS women, respectively.234 A link has been claimed between the degree of stromal vascularization and the response to gonadotropin stimulation in ART.371,372 According to Popovic-Todorovic and colleagues, ovarian power Doppler together with AFC are the two most significant predictors of ovarian response amongst 12 possible predictive factors investigated in normally cycling women.373 The observation of significantly lower 3D power Doppler indices of ovarian stromal blood flow

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in poor responders as compared to normal responders128 was taken as supporting evidence for the above. Studying the effect of age on ovarian stromal blood flow, Ng and colleagues observed that an age-related decrease was only observed in women 41 years of age or older.340a Using a 3D-based approach, Merce and colleagues found a correlation between ovarian VI and FI and the number of oocytes recovered.74 Vascular parameters, however, were not independent predictors of the number of follicles developed and oocytes retrieved; ovarian volume and AFC were, a finding consonant with other reports.5 Ng and colleagues observed that AFC, ovarian volume, and ovarian 3D Power Doppler flow indices did not significantly change after a short-term treatment with a GnRH agonist for pituitary down regulation.374 Järvelä and colleagues studied the power Doppler signal in the ovaries after pituitary suppression by GnRH agonist. They observed that Doppler data did not provide any additional information beyond that of ovarian volume

FIG. 35.29  A, Monitoring follicular growth in ART using the sonographic automated volume calculation worksheet. Each follicle and its measurements presented in the box at the lower right corner are coded with the same color for ease of interpretation. B, Vascularization of maturing ovarian follicles in COS induced for ART. In COS, starting 1 to 2 days prior to follicular maturity and hCG administration, color Doppler mode identifies the development of follicular vascularization. Characteristically, some but not all follicles display the development of end-follicular phase vascularization. (A From Ata B, Seyhan A, Reinblatt SL, et al. Comparison of automated and manual follicle monitoring in an unrestricted population of 100 women undergoing controlled ovarian stimulation for IVF. Hum Reprod 26[1]:127–133, 2011 Jan.)

and AFC in terms of predicting the subsequent response to gonadotropin stimulation during IVF.310

The Ovary in Polycystic Ovary Syndrome Echographic Characteristics of PCOS Ovary In the original description by Stein and Leventhal, PCOS implied verification of the characteristic ovarian morphology,375 a parameter which was later dropped.376,377 In 2003, the Rotterdam conference82 reinstituted an ovarian parameter in the diagnosis of PCOS when it included specific ovarian echographic criteria in the diagnostic criteria for PCOS. The Rotterdam consensus conference concluded that ovaries needed to hold more than 12 follicles of 2 to 9 mm each or measure more than 10 mL in volume to fulfill the PCOS criteria. In PCOS, a characteristic feature is an increase in stromal volume378,379 which in turn translates into an overall

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Allemand and colleagues389 questioned the number of small follicles that had been retained in the Rotterdam citeria for diagnosing PCOS, opting for a higher number of follicles or ovarian volume as cut off criteria. Using ROC curves, these authors proposed to set the diagnostic cut off at more than 20 follicles per-ovary, offering a predictive value of 100% and negative predictive value of 91%. Dewailly and colleagues contend that the small 2 to 5 mm follicles found in PCOS are actually atretic follicles that reflect follicular arrest,390 probably the result of an exaggerated inhibition of the terminal follicle growth.391 In PCOS, as in normally ovulating women, there is a strong correlation between AFC and AMH levels. Chen and colleagues observed that a significant correlation existed between the number of antral follicles and the ovarian volume on one hand and AMH on the other. AMH levels also correlated with total testosterone and the free androgen index.392 AMH levels were inversely correlated with body mass index (BMI), fueling the argument that the ovarian expression of PCOS is more profound in lean individuals.174 All findings indicate, however, that AMH levels parallel AFC and may do a better job defining PCOS and the degree of expression of this trait. AMH levels help when ultrasonographic data are not available.393

The Ovarian Stroma in PCOS B FIGURE 35.30  Corpus luteum appearance in gray-scale imaging (A) and color Doppler mode (B). On color Doppler mode, the characteristic intense vascularization of the corpus luteum confers the typical “ring-of-fire” appearance.

increase in ovarian volume.378 Ovarian suppression achieved with the OC pill results in reduced ovarian volume,315—a finding not shared by everyone380—but without changes in either AFC and AMH levels.353 Ovarian volume measurement raises methodological issues. Originally, 2D measurements of ovarian volume used the simplified formula for the prolate ellipsoid (0.5 × width × length × thickness), which assumes a regular ovoid shape of the ovary.381,382 Direct 3D measurements using the VOCAL-based rotation approach12,383 surpass the classical 3-diameter-and-ellipse-formula approaches.371 The novelty of the Rotterdam criteria is the introduction of echographic criteria in the definition of PCOS.384,385 However, the Rotterdam criteria have implicitly defined a category of women who do not qualify for PCOS diagnosis but have PCO-like ovaries, yet without androgen excess and the ovulatory dysfunction.386 If exposed to COH for ART, these women are at risk of ovarian hyperstimulation syndrome (OHSS), just like true PCOS women.174 Franks argued that the Rotterdam conference did not settle the controversy concerning the diagnosis of PCOS.387 He asserted that hyperandrogenism and anovulation are the mainstays of the definition of PCOS, while the Rotterdam criteria allowed for identification of PCOS women without clinical or biochemical androgen excess. Azziz stressed the fact that little is known about the degree of insulin resistance and long-term metabolical risk of oligo-anovulatory women with PCO ovaries, but without hyperandrogenism.388

The description of the echographic characteristics of the PCOS ovary goes beyond the cardinal features of an increase in size and number of small 2 to 10 mm follicles.394 An accessory echographic feature often put forth, yet more rarely objectively studied, is the increase in ovarian stromal echogenicity.395,396 Buckett and colleagues379 looked at the echographic appearance of 67 unselected women on no medication who were scheduled to undergo IVF. These authors measured total and stromal ovarian echogenicity, computing a stromal index as the ratio of stromal echogenicity over total echogenicity. Criteria for PCOS, AFC greater than 10, were found in 37% of the cases. Total ovarian volume and stromal volume were higher in PCOS ovaries, but contrary to expectation, there were no differences in mean stromal echogenicity between PCOS women and their normal cycling counterparts. There was, however, a reduction in mean echogenicity resulting from the increase in ovarian follicles, which accounted for a significant increase in stromal index in spite of the lack of change in stromal echogenicity per se. These results were confirmed with the most recent VOCAL-based volume measurements.397 Lam and colleagues compared 40 Caucasian women identified as having PCOS by the Rotterdam criteria and 40 cycling controls having normal ovaries on ultrasound.397a Ovarian and stromal volumes were markedly larger in the PCO group (12.56 and 10.79 mL) as compared to controls (5.66 and 4.69 mL), but there was no significant difference in stromal echogenicity between PCOS and controls, respectively. Likewise, Jarvela and colleagues398 failed to find a difference in echogenicity between 14 PCOS and 28 women with echographically normal ovaries. In subgroup analyses, Lam and colleagues observed that hirsute PCOS patients had larger stromal volume than the nonhirsute counterparts,397a but found no differences in ovarian stroma echogenicity between these subgroups. It can be concluded, therefore, that the impression of denser ovarian stroma in

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PCOS results from a visual illusion linked to the increased stromal volume and a decreased echogenicity of the periphery of PCOS ovaries due to the increase in follicles. Interestingly, AFC remains constant in PCOS women taking OC pills in spite of the profound decrease in ovarian volume.316 This indicates that the effect of hormonal contraception is on ovaran stroma not follicles.399 The availability of pulsed and color Doppler functions on vaginal ultrasound probes led to the study of intra-ovarian stromal flow as a distinct phenomenon from changes in flow taking place in the follicular walls,400 and after ovulation in the CL.355,401,402 Stromal flow was found to be increased in women suffering from PCOS371 as compared to findings in cycling controls.130,136 Zaidi and colleagues compared ovarian stromal flow status measured by Doppler (peak systolic blood flow velocity) to ovarian responsiveness to FSH and hMG in ART cycles. Their observation found a positive correlation between blood flow at baseline measured by Doppler and ovarian response to COH with higher values in 13 POCS patients as compared to 63 cycling controls.403 Conversely, ovarian blood flow was decreased at baseline in women found to have a poor response as compared to normal responders.371 In 2D-based studies, Balen and colleagues concluded that there is an increase in ovarian blood flow in PCOS,386 a finding shared by some288 but not by other investigators. Using 3D PDA, Jarvela and colleagues failed to find an increase in stromal vascularity in 14 women with PCOS as compared to 28 control women.398 Lam and colleagues88 compared ovarian flow assessed by 3D ultrasound in 40 women fulfilling the Rotterdam criteria for PCOS with those of 40 age-matched cycling controls. Ultrasound data were obtained on day 3 to 5 after spontaneous or induced bleeding. A 3D-based data set was acquired using a sweep angle set to 90 degrees and a slow-sweep mode. Per ovary AFC was significantly higher at 16.3; 9 to 35 (median; range) in the polycystic ovary group as compared to controls (5.5; 2 to 10). The results showed an increase in some (VI) but not in all 3D-PDA indices (FI). Interestingly, differences were observed among various phenotypes of PCOS. Ovarian blood flow was increased in a subgroup of 30 of 40 hirsute PCOS women as compared to their 10 nonhirsute PCOS counterparts. Likewise, blood flow was also higher in 14 lean PCOS women as compared to their overweight counterparts. Yet, there were no differences in AFC between lean and obese PCOS and hirsute and nonhirsute women. Ng and colleagues403a confirmed the differences in ovarian blood flow between different PCOS phenotypes. This team also found a significant increase in ovarian blood flow in women whose BMI was less than 25 as compared to their overweight counterparts with a significant negative correlation between total ovarian indices (VI, FI, and VFI) and BMI.174 There was also a strong trend toward higher LH levels in lean PCOS women. Hirsute women with PCOS had increased stromal volume compared to nonhirsute women with PCOS.404

Early Diagnosis of PCOS in Adolescents and Prepubertal Girls Chang and Coffler stressed the importance of diagnosing PCOS early in adolescent girls in order to offer treatments for the hormonal (hyperandrogenism and hirsutism) and

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metabolical expression of the disease.298 Based on studying AMH levels through the pubertal process, Crisosto and colleagues observed that the increase in follicular mass is established during early development and persists during puberty.405 First amongst individuals in whom an early diagnosis of PCOS is important are daughters of PCOS women. Sir-Petermann and colleagues determined that the daughters of women with PCOS are at increased risk of suffering from PCOS themselves.406 These authors compared 14 female infants (2 to 3 months old) and 25 prepubertal girls (4 to 7 years old) born to PCOS mothers to a control group of 21 female infants and 24 prepubertal girls born to mothers with regular menses and without hyperandrogenism. Serum AMH levels were significantly higher in PCOS as compared to the control group during early infancy (20.4 ± 15.6 versus 9.16 ± 8.6 pmol/L; P = 0.024) and during childhood (14.8 ± 7.7 versus 9.61 ± 4.4 pmol/L; P = 0.007). Yet, gonadotropin and serum sex steroid concentrations were similar in both groups during the two study periods, except for FSH, which was lower during childhood in girls born to PCOS mothers. The discrepancy between the alteration of the follicular cohort, as expressed by the increase in AMH levels, and the lack of hormonal alteration in these infants and prepubertal girls suggests that the ovarian facet of PCOS precedes the altered gonadotropin pattern. That there is evidence of an altered follicular development during infancy and childhood speaks for the need of screening ovarian patterns either directly by ultrasound or through the surrogate for the follicular cohort, AMH levels.

Adnexal Cysts and Tumors Ovarian versus Extra Ovarian Pathologies Vaginal ultrasound has become the primary approach used for investigating adnexal masses. As stressed in a review by Brown290 the primary question that pelvic imaging needs to answer is whether the adnexial mass is of ovarian or extraovarian origin. Most adnexal masses arise from the ovary. When the mass is of ovarian origin, malignancy is a concern. Once established that the mass is of extraovarian origin, short of rare cases of tubal cancer,407 the mass is not likely of cancerous nature. The primary clinical presentation of tubal cancer is most commonly one of pelvic pain rather than an asymptomatic pelvic mass.408 If the ovary and the tube are both involved with tumor, the bulk of the tumor should be in the tube.409 Identifying small antral follicles can help determine whether a mass is ovarian or not. Likewise, recognizing an ipsilateral ovary independent from the adnexal mass allows for recognition that the mass is nonovarian in nature. In postmenopausal women, the absence of visible ovarian follicles complicates ascertainment. Helpful tips for determining the ovarian or nonovarian nature of the mass include the possibility of mobilizing the structure that is being investigated by pressure applied with the vaginal probe. In case of a cystic mass, the impact of the pressure applied with the probe on the cystic dilatation should be followed. In case of an ovarian cyst, the probe usually bounces off the nondeformable cyst wall. When the cystic structure is a hydrosalpynx, the pressure of the probe will modify the shape of the cystic dilatation as fluid displaced by the probe will move elsewhere along the length of the tube.

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The vascular supply of the lesion may also help in delineating the ovarian or extra-ovarian origin. In the case of pedunculated fibroids, one might identify vessels bridging with the uterus. The peripheral development of vessels can also help in recognizing the fibroid nature of an adnexal mass.

Extra-Ovarian Pathology Paraovarian (or paratubal) cysts appear as simple cysts indistinguishable from ovarian cysts, if it were not for their nonovarian nature, which can normally be determined in premenopausal women because ovarian tissue can easily be identified,410,411 including identification of an ipsilateral ovary.412 Hydrosalpinges are the most important para-ovarian structures encountered in clinical reproductive endocrinology. Typically, they appear as an elongated cystic mass.413 Other typical features of hydrosalpinges include the waist sign and images evoking beads on a string. In a review of 67 cystic adnexal masses, Patel and colleagues observed hydrosalpinges in 26 (39%). These authors concluded that an elongated aspect, together with the waist sign, carried the highest probability for hydrosalpinx.414 Ultrasound scans aimed at excluding the presence of hydrosalpinges in ART need to be performed at midcycle because fluid tends to accumulate during the follicular phase or after ovarian stimulation,415 including in women with PCOS.416 Peritoneal inclusion or pseudocysts are believed to result from fluid trapped within pelvic adhesions which may create the impression that the ovary lies within the cyst itself.290,417

Ovarian Cysts and Tumors Brown described five primary findings for ovarian masses, each having their echographic characteristics, which most often permit a positive diagnosis.290

Simple Cysts In premenopausal ovaries, a simple cyst is a structure greater than 3 cm in diameter with a thin anechoic wall free of protrusion and showing characteristic distal enhancement. Structures of lesser diameter are best described as follicles, which may have stopped developing or failed to regress because of isolated or recurrent ovarian dysfunction. Simple cysts most often disappear spontaneously, a process that may be hastened by suppressing ovarian function with the OC pills. The majority of persisting cysts treated surgically turned out to be serous cystadenomas.

The Corpus Luteum The imaging characteristics of the CL may be so dramatic that they can easily be mistaken for cancer because of the intense low impedance blood flow. Baerwald and colleagues followed luteal images and serum E2 and progesterone in 50 women daily from one ovulatory episode to the next.311 The day of ovulation was defined as the day of follicle disappearance. The CL was detected on the day of ovulation and later in 100% of women, and during the subsequent follicular phase in 90% of them. Some CL presented as a central fluid filled cavity (identified in 88%), and some without (12%). A progressive decrease in echogenicity was observed throughout the luteal phase. The CL is intensely vascularized with an annular arrangement of vessels around the CL

itself, which can be recognized on gray-scale images by a circular dual contour line.132

Hemorrhagic Cysts Hemorrhagic cysts are common occurrences in premenopausal women. They can be associated with acute pain or they can constitute a fortuitous finding in routine scans done prior to ART. The internal echo pattern varies depending on the stage of the hemorrhage and the amount of fluid present. The acutely hemorrhagic cyst is typically more echogenic than the surrounding ovarian tissue. Characteristically, findings will evolve first to a sponge-like appearance, later followed by the development of irregular internal echos after the clot starts to retract after a few days, leaving a mesh-like maze of intertwined fine structures that are the echographic expression of fibrin strands.418,419 These rarely run totally across the cyst. A gentle brisk pressure stroke applied with the vaginal probe may generate a disruption wave that propagates through the mesh-like network of internal echos with a characteristic jello-like effect where the wave rebounds backwards after hitting the cyst wall. The retracted blood clot may mimic a mural nodule. The sharp angles of the retracting clot are different from solid tumors that do not tend to have acute angles.419 To distinguish the clot from more ominous internal echos, it is most helpful to document the absence of vessels entering the solid area. MRI is classically of little help for diagnosing hemorrhagic cysts.420 Most commonly, hemorrhagic cysts rapidly change their echographic appearance and ultimately disappear, a process that may be hastened by suppressing ovarian function with OC pills.421,422 The pill is commonly started on day 2 of the menstrual cycle or arbitrarily, provided that endogenous progesterone is low (less than 1.5 ng/mL), which precludes the possibility of pregnancy. The differential diagnoses include endometrioma and dermoid cysts. Typically, the former has a homogeneous mixed echogenicity (gray) appearance, whereas the latter may add to that grayish appearance the finding of brightly echogenic calcifications, and on occasion, a denser mesh image when the dermoid cyst contains an accumulation of hair. Occassionally, a hemorrhagic CL may have a clinical course suggestive of ectopic pregnancy.

Endometriomas Unlike other locations of endometrisosis which are difficult to identify, ultrasound examinations are efficient for diagnosing ovarian endometriosis, the endometriotic cyst or endometrioma. Out of 1170 scans yielding 252 adnexal masses the diagnosis of endometrioma was made in 40 cases (prevalence 16%).423 Taken in isolation, low level internal echoes have a sensitivity of 93% and a specificity of 83%. At the other end of the spectrum, identifying all characteristic findings of endometriomas, namely low-level internal echoes, no neoplastic features, and hyperechoic wall foci or multilocularity, led to a specificity of 99%, whereas, sensitivity dropped to 45%. These data demonstrate that gray-scale vaginal ultrasounds can achieve a high degree of accuracy for diagnosing endometriomas. Hence, an adnexal mass with diffuse low-level internal echoes and no neoplastic features is most likely to be an endometrioma.

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Mature Cystic Terratomas or Dermoid Cysts Typical features of dermoid cysts include shadowing echo­ density, regional diffuse bright hyperechoic areas with an attenuating effect, hyperechoic lines and dots and fat-fluid levels.424 Because of the great variability in the appearance of cystic terratomas has been emphasized, it has been suggested that MRI is needed to rule out the possibility of ovarian cancer, except when the most typical echographic are observed. Suspecting that echographic findings are sufficiently specific, Patel and colleagues demonstrated in a prospective study that 55 of 74 cystic terratomas diagnosed amongst 252 adnexal masses had two or more sonographic features associated with dermoids.425 These authors observed a positive predictive value of 80% for shadowing echogenicity, 75% for regionally bright echoes, 50% for hyperechoic lines and dots, and 20% for fat-fluid level. Taking these signs together, each reviewer had 98% positive predictive value, which reached 100% when an adnexal mass had two or more sonographic findings associated with dermoids. Most ovarian cancers are epithelial neoplasms, which include serous, mucinous, endometrioid, and clear cell neoplasms. Additionally there are borderline-malignancy tumors or tumors of low malignant potential of all epithelial cell types with the mucinous and serous cell types being by far the most common. The presence of a solid component is the most common feature that reveals the cancerous nature of an ovarian tumor. In spite of the initial interest in color and power Doppler, there is a consensus that Doppler indices do not provide much more information than grey scale morphological assessment of suspected ovarian tumors.

Contrast Imaging of the Fallopian Tubes The fallopian tubes are usually not identified on pelvic ultrasound, nor with other imaging approaches such as MRI or CT scans. Dilated tubes in case of hydrosalpinges are easily seen, however, by the contrast effect generated by fluid contained in the tubes. Exploring the fallopian tubes, therefore, requires some sort of contrast. Classically, the fallopian tubes are visualized on x-ray images by transcervical infusion of an opaque dye in HSG. Positive (white) contrast media are routinely used.426

Hysterosalpingography Indications Hysterosalpingography (HSG) is the ancestral imaging tool used for investigating infertility.427,428 The primacy of HSG for exploration of the uterine cavity is challenged by transvaginal ultrasound and HySo on one hand, and hysteroscopy on the other. The former offers the advantage of depicting both the cavity and the myometrial contour. This made HySo the undisputed primary imaging tool for investigating uterine malformations. The miniaturization of endoscopic instruments has allowed diagnostic hysteroscopy to become an office procedure, thereby leading to marked increases in its use. Diagnostic hysteroscopy offers the advantage of direct visualization of the pathological structures. Sonography using positive contrast products allows delineation of the tubal lumen, which appears white on grey scale images. Positive contrast agents

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used include Echovist®, Levovist®, and Albumex®, none of which is approved for use as fallopian tube contrast medium by the Food and Drug Administration (FDA).429 The need for HSG, traditionally included in all infertility workups, has been questioned for women whose serology for Chlamydia is negative. In these women, it has been claimed that HSG has a yield of tubal pathology that is so low that the routine use of HSG is not cost-effective.430,431

Technical Considerations Classically, HSG is performed during a relatively short interval between cycle day 5 and days 12 to 14. This time limit achieves two objectives: (1) menses must be finished in order to minimize the risk of enhancing retrograde bleeding by the procedure; (2) The x-ray procedure is performed before ovulation in order to minimize the risk of irradiating a developing pregnancy. It may be handy to place the patient on a short course of OC pills for the sake of programming. Several thorough reviews on HSG have been published which describe in detail practical aspects of this procedure.432-434 Many groups routinely prescribe 600 mg of ibuprofen or other nonsteroidal anti-inflammatory drugs (NSAIDs) 30 minutes to 1 hour before the procedure in a effort to ease the uterine cramping that may be generated by the procedure.433 Frishman and colleagues randomly compared the effects of intrauterine instillation of 2% lidocaine solution or saline before performing an HSG.435 Moreover, Zhu and colleagues reported less pain after warming the contrast medium.436 The issue of routine antibiotic prophylaxis has been much debated, with infectious complications being less than 1%. With the patient in the dorsal lithotomy position and a speculum in place, the instillation device is positioned in order to provide a sealed connection with the cervix. The instillation instrument is either a Jarcho canula,437 which is held to the cervix with tenaculums, or plastic catheters equipped with inflatable balloons, such as the 5-F HSG catheter (Cooper Surgical, Trumbull CT). The balloon is inflated once it has passed the internal os of the cervix. The balloon must be filled with the same contrast material as used for the examination. While the latter are probably easier to handle, they may create pain because of the balloon,438 and in certain circumstances, may hinder the vision of the cervical canal. The speculum needs to be removed once the instillation instruments are in place. Air needs to be meticulously expelled from the instilling device so that only the contrast medium is pushed into the uterine cavity. Failing to do so could lead to a false diagnoses of filling defects. Prior to instilling contrast medium, and after a mark identifies sides, a scout x-ray of the pelvic cavity is examined, looking for possible calcifications. Water-soluble contrast material is slowly instilled under intermittent control on fluoroscopic vision. A minimum of four spot radiographs are recommended.434 The first image is obtained during early filling of the uterine cavity in search for filling defects, which may later be obscured when the uterus is totally opacified. The second image is obtained when the uterus appears fully distended. The third image aims at depicting the fallopian tubes. A fourth image is obtained to detect free spillage of dye into the pelvic cavity and proper mixing. Additional spot radiographs are obtained as necessary, including oblique views to avoid the superimposition of images.

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Choice of Contrast Medium: Oil-Based or Water Soluble? The original contrast media used in HSG was lipid based, with Lipiodol being used since it became available in the early 1920s.439 In a pre-IVF era study, Mackey and colleagues compared the occurrence of spontaneous pregnancies when women had an HSG or not, sorting out results for when the oil-based Ethiodol or a water-soluble contrast medium was used.440 In their study population of 460 women, the spontaneous pregnancy rate over 1 year following oil-based dye HSG was 58%, whereas it was 38% in women in whom a water-soluble dye was used. These puzzling results, which speak in favor of a fertility enhancing effect of HSG using lipid-based dye as compared to findings observed following HSG using water-soluble dyes, were confirmed in a number of other studies.441-443 Oil-based contrast medium come with the risk of granulomas,441 or the serious complications of a pulmonary or cerebral oil embolism.444 A sequential combination of water-soluble dye for documenting tubal permeability followed later by instillation of oil-based Ethiodol has been proposed to benefit from the fertility enhancing effects of HSG using oil-based dyes while avoiding its risk. Alternatively, the use of the oil-based dye has been proposed in women whose tubal patency had been demonstrated by laparoscopy.445 Water-soluble dyes offer better visualization of the distal regions of the fallopian tubes, allowing identification of striae.443,434 Attempting to provide an explanation for the pregnancy promoting properties of oil-based dye HSG, Goodman and colleagues proposed that the lipid dye inhibited the activity of peritoneal lymphocytes and macrophages.446 That HSG was found to enhance fecundity in women whose infertility was of unknown etiology,441 or due to endometriosis,374 suggests that the inhibiting effect of the lipid dye on macrophages might be most significant in this subset of patients.447

The Uterine Cavity Filling defects constitute a common finding in HSG. Once artifacts such as air bubbles or endometrial folds have been excluded, the differential diagnosis is synechiae as a result of post-traumatic or infectious endometrial scarring. Full fledge development of endometrial scarring leads to Asherman syndrome, classically associated with multiple scarring of the endometrium and clinically, amenorrhea. Myomas may indent the uterine cavity. For practical purposes, the degree of distortion must be assessed to determine whether surgery is indicated in the absence of symptoms, and whether an intracavitary approach is feasible. HySo allows for simultaneous visualization of the whole fibroid and the portion that extends into the uterine cavity. Assessment of the uterine cavity includes a search for congenital malformation of the organ. 3D sonography— with or without HySo—has taken the lead for diagnosing uterine malformations by offering simultaneous vision of the uterine cavity dilated by saline and the organ itself identified on gray-scale imaging.448 The 3D reconstruction of the uterus in the frontal plane is of great practical help,449 and even lends itself to virtual hysteroscopy exploration450 (Fig. 35.31). Finally, the assessment of the uterus must include the cervical canal, notably to rule out cervical insufficiency.

Clues regarding the possibility of adenomyosis can arise from HSG images.451 The characteristic image is one of diverticula filled with contrast medium that extends into the myometrium. This may exist in the context of an overall irregular contour with multiple outpouchings. The ultimate expression is the classical honeycomb pattern.452,453 Although it is held as being diagnostic of adenomyosis, it is now recognized as not being specific.451,454 In cases of large adenomyomas, the image may be one of a mass-like filling defect, with contrast material filling the mass. Adenomyosis is more accurately diagnosed by MRI where it is characterized by a thickening of the junctional zone.455 This is the only feature that has been fully validated against histological identification of adenomayosis in women undergoing hysterectomy.

The Fallopian Tubes In spite of emerging approaches based on positive contrast enhanced ultrasound imaging, HSG remains the best method for visualizing and evaluating the fallopian tubes. Normal tubes appear thin, with smooth outlines and a characteristic distension in the ampullary area. Proximal disease that prevents the visualization of the tubes may be the reflection of salpingitis isthmica nodosa (SIN) or the result of a simple spasm. In the case of SIN, the penetration of the contrast medium into the thickened fallopian tube wall produces the typical honeycomb appearance (Fig. 35.32). SIN is usually the result of pelvic inflammatory disease that leads to fibrosis of the proximal tubal section with stricture of the lumen and tubal occlusion.456 We now know that SIN may also result from a tubal form of endometriosis.456 In this case, temporary treatment with a GnRH agonist has been documented to be effective,457 offering alternatives to the surgical approach for SIN.458 Our experience showed that 3 months of treatment with GnRH antagonist resulted in repermeabilization of 15 of 18 women who had bilateral proximal occlusion, and in whom a laparoscopy showed some endometriosis and no evidence of distal tubal disease.73 We believe that medical treatment of SIN should be considered each time endometriosis is present and there is no evidence of distal tubal disease. Spastic constriction of the tube may result in complete tubal occlusion, opening the door to an erroneous diagnosis of proximal tubal disease. Timely administration of glucagon promotes uterine muscle relaxation and can be used to reverse spasms.459 Distal disease is usually associated with dilatation of the tube as a consequence of hydrosalpinges or the incomplete form, sactosalpinges. Pelvic inflammatory disease is the most common cause of distal tubal disease, but it may at times result from endometriosis.460,461 We believe that the diagnosis of hydrosalpinx should be made on visualizing dilated tubes on ultrasound at midcycle. This nuance is important, as it is commonly held that hydrosalpinges must be removed (salpingectomy) to optimize IVF outcome.44 From the clinical trials that exist, there is now a consensus for recognizing that pregnancy rates in IVF are approximately halved when a hydrosalpynx is visible on ultrasound.336,462 Taylor’s group found that hydrosalpinges, probably by the release of fluid into the uterine cavity, interferes with the role of HOXA10 in receptivity and implantation.44,463-465

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A

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B

C FIGURE 35.31  Hysterosalpingography (HSG) showing a completely unfused uterus with two cervices and two corpora uteri. Note right (A) and left (B) uterus and tube. Corresponding appearance on magnetic resonance imaging (MRI) (C). (From Imaoka I, Wada A, Matsuo M, et al. MR imaging of disorders associated with female infertility: use in diagnosis, treatment, and management. RadioGraphics 23:1401, 2003.)

Complications

FIGURE 35.32  Characteristic honeycomb appearance in the proximal segment of the fallopian tube seen in case of salpingitis isthmica nodosa (SIN).

Minor complications of HSG include pain and cramping, which is usually mild to moderate and subsides within hours of the procedure. The most serious complication is a pelvic infection. Albeit rare (less than 1%), the risk exists, particularly in cases with a past history of pelvic inflammatory disease.466 Some have advocated for antibiotic prophylaxis, either systematically or in selected cases in which there is a higher risk of infection.467 The most common regimen has been doxycycline 100 mg, twice a day, administered for 1.5 to 5 days. We commonly administer 100 mg immediately prior to the procedure, on the evening of the procedure, and the morning after the procedure in women at higher risk of infection by history or findings on HSG. Other infrequent complications include allergic reaction to iodine based contrast agents. In the latter case, gadolinium can be used as a replacement.468 Perforation of the uterus or fallopian tube has been reported with HSG, but it is extremely rare.

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Infusion Sonography Numerous publications have described the possibility of documenting tubal patency by infusion sonography.469 A consensus has been reached over the fact that positive contrast solutions providing white visualization offer better and easier visualization of the tubes, particularly of the proximal tubal sections.429,470 Solutions such as Echovist® or Levovist®, while offering unquestionable value for assessing tubal patency suffer from: (1) Not providing an image of the fallopian tubes that matches that of HSG, particularly of the distal tubal segment; (2) the not insignificant cost of the contrast product. 3D reconstruction capability is likely to facilitate tubal assessment by sonography, but without inherent changes to imaging capabilities.471 It is our impression that while the proximal section of the fallopian tube can easily be identified, HSG images remain largely superior for assessing the distal ends of the tubes. A key issue when attempting to assess tubal physiology is to determine whether retrograde transport of sperm takes place normally during the late follicular phase. Using a 99Tclabeled macroalbumin aggregates in a procedure referred as hysterosalpingo scintigraphy, Kissler and colleagues reported evidence of retrograde transport alterations in endometriosis and adenomyosis.472,473 This approach is cumbersome, however, and does not lend itself to repeated measurements, nor to studies conducted in mock reproductive conditions. An advantage of HySo over x-ray-based methods for assessing tubal function is the possibility of repeating examinations. Unfortunately, the ideal infusion preparation for assessing tubal function does not yet exist. An ideal preparation capable of fulfilling the expectations of infertility specialists would be a product that would have the following characteristics: (1) offer positive contrast imaging for identification of the fallopian tubes, (2) a consistency that permits lasting contrast properties for durations sufficient for studying whether actual transport takes place through the tubes, (3) nontoxicity to gametes. This latter characteristic would be helpful for studying retrograde tubal transport under intercourse-like conditions, when the uterotubal unit is exposed to prostaglandins and other constituents of semen. This is deemed necessary as constituents of semen may exert clinically relevant effects on retrograde contractility474 or other uterine functions.475 Uterine and tubal influences exerted by semen constituents may be facilitated by a direct vagina-to-uterus transport through the functional portal system, or first uterine pass effect.476

Endometriosis The clinical expression of endometriosis is infertility477 and pelvic pain,478 which is notoriously exacerbated in cases with endometriomas.479,480 One can distinguish the manifestations of endometriosis that are readily identifiable on ultrasound (i.e., endometriomas) from others that are known to evade conventional vaginal ultrasound scrutiny. Identifying and staging endometriosis, and its possible deep pelvic infiltration, has been the primary basis for reverting to MRI in benign gynecology imaging.481,482 In certain circumstances, rectal ultrasounds or special procedures such as sonovaginography199 have been proposed for evaluating the presence and extension of deep infiltrating endometriosis. Preoperative staging is challenging, and requires

exploring all the possible locations of endometriosis. Vaginal ultrasound, put in expert hands, is now competing with MRI to the point that it may become the first-line diagnostic tool.483 While endometriosis can affect all organs and structures of the pelvic cavity, its extension commonly follows known paths. It is widely accepted the ovary constitutes the most common site for the development of endometriosis, where endometriotic cysts or endometrioma are formed. In general, peritoneal lesions are classified as either superficial or deep, depending on whether penetration exceeds 5 mm or not. Anterior endometriosis includes invasion of the bladder, and particularly its detrusor muscle.484 Posterior endometriosis includes the extension of the disease to the uterosacral ligament, the upper and posterior portion of the cervix (torus) and finally, the various forms of vaginal, bowel, and uretral endometriosis.485,486

Endometriotic Cysts (Endometriomas) Endometriomas are best diagnosed and described on ultrasound, where they present as isolated or multiple cystic structures filled with homogenous low-level echos.418,487 This gives what is commonly referred to as grayish fairly homogenous aspect to the cystic structure. The majority of endometriomas range from 30 to 60 mm.487 Color Doppler helps establish the endometriotic nature of newly identified cysts, and can document the absence of blood vessels within the walls of the cyst, or penetrating inside the cyst.485 Differential diagnosis includes benign teratomas425 and hemorrhagic cysts, particularly CL cysts.488 Benign teratomas because of their sebum content, which has the general consistency of the old blood found in endometriomas, may provide echographic characteristics that are fairly similar to those of endometriomas. The presence of echos of hairballs, or shadowing effects from calcified structures (bones), strongly speaks for a dermoid cyst.489 Hemorrhagic CL cysts vary in size (2.5 to 10 cm). They differ from endometriomas by the presence of complex internal architecture that reflects the presence of a retracted clot and its fibrin content,419,490 a characteristic that can be appreciated with new automatic texture assessment.491 Moreover, a sponge-like reticular pattern is often identified within the cyst.365 These two findings are not encountered in endometriomas. The ultimate hemorrhagic nature of cysts is confirmed by witnessing their disappearance (or profound change) on repeat ultrasounds performed 2 to 4 weeks later, a measure mandatory in our eyes.492 Endometriomas are often multiple. The choice of surgical management or ART will be influenced by the unilateral or bilateral nature of the lesions.493-495 They characteristically have hyperchoic wall foci that are clearly identified in nearly a third of the cases.423 In a recent study conducted on 65 women suspected of having an endometrioma, Alcazar and colleagues observed that peripheral vascularization of the endometrioma’s wall was more important when pelvic pain was present than when it was not.496 Although ultrasound is the diagnostic tool of choice for exploring endometriomas, in certain circumstances, MRI may prove to be more helpful, let alone necessary when ultrasound findings are inconclusive or their interpretation is hampered by the presence of other disorders, notably uterine fibroids.497,498

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Endometriosis of Pelvic Areas Other Than the Ovary Deep endometriosis is defined by lesions that extend ≥5 mm beneath the surface of the endometrium499 and are associated with increased nerve density.500 Chapron and colleagues stressed the fact that pelvic examination does not suffice for reliably diagnosing and locating deep endometriosis prior to surgery.288 While elements in the patient’s history,500 particularly at the time of adolescence,500 such as an early use of OC pills for managing severe dysmenorrhea,500 or phenotypic characteristics,501 increase the risk of finding endometriosis, these alone or collectively do not suffice for making the diagnosis preoperatively.502 Taken together, these elements mandate deploying an image-based workup aimed at diagnosing and mapping deep endometriosis,288 using diagnostic criteria,503,504 before undertaking surgery. While MRI remains the gold standard for diagnosing deep endometriosis preoperatively, vaginal ultrasound is becoming ever more efficient.502,505,506 Deep endometriosis lesions are described as hypoechoic linear thickenings or nodular masses with and without regular contour in the cul-de-sac, retro-cervical region, and recto-vaginal septum, involving or not involving the vaginal wall.502,505 Several groups stress the fact that vaginal ultrasound allows the visual identification of lesions to be correlated with pain generated by exerting pressure on the suspected lesions.507,508 Abrao and colleagues found that transvaginal ultrasound offered better sensitivity and specificity for identifying deep endometriosis than pelvic examination and even MRI.509 The fact that all patients had an enema prior to the ultrasound examination might have contributed to the positive findings. Dessole and colleagues underscore that saline placed in the vagina can improve the visualization of the vaginal wall.199 Anterior and bladder involvement of endometriosis is an extension that is found in nearly 20% of endometriosis cases.510,511 Bladder endometriosis commonly declares itself by localized bladder wall thickening on ultrasound.512,513 At times infiltration may be recognized by the presence of deep irregular hypoechogenic infiltration of the bladder wall, particularly in the area of the uterobladder flap, with protrusions that may extend inside the bladder.288 At MRI, bladder lesions are identified as heterogeneous T2 isointense thickening of the bladder wall,485 with lesions varying from 10 to 40 mm.504 In a study involving 195 patients, MRI had a sensitivity and specificity of 88% and 99%, respectively, for the diagnosis of bladder endometriosis.481 Extension of endometriosis to the ureter can be identified on T2weighted sequences.485,514 Endometriosis of the uterosacral ligament and posterior aspect of the cervix could be assessed by transvaginal ultrasound in 64% of cases, a diagnosis confirmed surgically in 88% of cases.481 MRI provides higher sensitivity, however, with reported values ranging from 76% to 86%.481,504 Retrocervical endometriosis is identified when thick irregular nodular formations of hypoechoic nature are found in this area, with possible extension to one or both uterosacral ligaments. Signs of adhesion are looked for by sliding loops of bowel along the uterosacral ligament and retrocervical area. The sensitivity of ultrasound is notoriously weak, however, reaching 64% in Bazot’s hands481 for diagnosing retrocervical endometriosis. In contrast, MRI offers an improved

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sensitivity for diagnosing deep endometriosis of the retrocervix.504 An elegant MRI-based study demonstrated that deep infiltrating endometriosis does not originate from the rectovaginal septum, but rather from the posterior of the cervix at the height of insertion of the uterosacral ligaments.288 Endometriosis of the bowel most often, but not exclusively, affects the rectosigmoid colon,515 a finding in agreement with the intriguing, yet generally recognized assymetry of pelvic endometriosis.516-518 It is characterized by long, nodular, predominantly solid hypoechogenic lesions with varying degrees of infiltration from the serosal layer down to the muscularis propria identified as two hypoechoic lines separated by a fine hyperechoic line.502,509,514,519 On MRI, the reported sensitivity and specificity is of 84% and 99%, respectively, in women with documented intestinal involvement.481 Diagnostic criteria for rectal invasion include rectal wall thickening and a characteristic triangular attraction of the rectum toward the posterior aspect of the cervix or torus uteri.481,485,512,520 Preoperative staging of endometriosis extension, and particularly, identifying the presence and extent of deep lesions, is considered an obligation today.521

Adenomyosis A variant of endometriosis, adenomyosis, is characterized by the heterotopic development of glands and stroma in the subendometrial myometrium with various degree of hyperplasia present amongst adjacent smooth muscle cells.522 Adenomyosis can be either diffuse or focal.523 In the diffuse form, glandular and stromal constituents of the endometrium extend throughout the sub-endometrial layers of the myometrium, resulting in an overall enlargement of the whole uterus.522 In the localized variant, the affected area is isolated in the frontal or posterior wall of the uterus, taking on an appearance that may be difficult to distinguish from a fibroid.524 While adenomyosis has been claimed to cause infertility, its contribution is debated.455,525 Various contemporary transvaginal ultrasound approaches are being tested and developed to identify and delineate the territorial extension of adenomyosis. The most commonly identified marker of diffuse adenomyosis on ultrasound is a poorly marginated hypoechoic extension in the subendometrial layers of the myometrium.526 According to Devlieger and colleagues, localized adenomyosis can be distinguished from fibroids based on the following criteria: (1) absence of circular vascularization at the border of the lesion, which may be replaced by vessels actually penetrating into the lesion; (2) presence of a shaggy limit between the lesion and the outer myometrium that typically lacks the characteristic shell common to fibroids with its acoustic shadowing effect (Fig 35.33).524 MRI, especially through T2-weighted images, provides excellent soft tissue differentiation. In the uterus, MRI delineates a subendometrial band of low-signal referred to as the junctional zone (JZ). Histological analyses have demonstrated that JZ corresponds to the subendometrial layer of the myometrium.527,528 Yet, these studies failed to provide clues as to the nature of the histological basis that accounts for the low-signal on MRI. In contrast to the outer layers of the myometrium, the subendometrial layer of the myometrium is of müllerian origin and the site of changes

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A FIGURE 35.34  Endometriotic lesion: Sagittal T2-weighted fast spinecho magnetic resonance imaging (MRI) of the pelvis showing an infiltrative lesion (typical fibromuscular lesions of endometriosis) extending to the anterior rectal wall and posterior wall of the uterus (arrow).

B FIGURE 35.33  Diffuse adenomyosis. A, The ultrasound appearance is characterized by a thickened subendometrial sonoluscent area. B, Magnetic resonance imaging (MRI) is characterized by a diffuse and fairly regular thickened subendometrial transitional zone.

in E2 and progesterone receptors that follows the pattern of changes encountered in the menstrual cycle.26 Considerable variation in JZ thickness has been observed, with reported values that range from 2 to 8 mm.529,530 In one report, transvaginal ultrasound was as effective as MRI in delineating the subendometrial layer of the myometrium.531 Excessive local or diffuse thickening of the low-signal JZ with ill-defined boundaries has been the hallmark of adenomyosis, with many authors proposing 8 mm as the cutoff value between normal and overt diffuse adenomyosis.532 In a study of 119 patients undergoing hysterectomy, the JZ was 7.7 mm in 91 women without adenomyosis, and 15 mm in 28 women with adenomyosis.531 In women whose JZ was measured between 8 and 12 mm, associated findings such as focal thickening of the JZ or high-signal foci within areas of low signal on T2-weighted sequences may represent island of ectopic endometrium.

Focal adenomyosis in areas of low-signal intensity on T2weighted images was found to consist of smooth muscle hyperplasia associated with heterotopic endometrial tissue (Fig. 35.34).454,533 It is important to distinguish focal adenomyosis from leiomyomas. In 21 women suspected to have focal adenomyosis, MRI adequately diagnosed signs of focal adenomyosis in 12 women. In 10 of these women, the diagnosis was ultimately confirmed by histological analysis of the pathological specimen.534 According to Reinhold and colleagues,454 features that favor the diagnosis of adenomyosis on MRI include: (1) lesions with poorly defined borders; (2) lesions that extend along the endometrium; (3) minimal mass effect on the endometrium relative to the size of the lesion; (4) linear striation radiating out of the endometrium into the myometrium; and (5) absence of large vessels at the margins of lesions contrary to the typical findings of leiomyomas. Furthermore, these authors stress the possibility that uterine contractions and the resulting deformation of the endometrialmyometrial interphase may generate images evocative of focal adenomyosis.454 There is general agreement about the high sensitivity of MRI for detecting focal adenomyosis, with sensitivities of 88% and 86%, reported respectively, in studies by Ascher and colleagues532 and Reinhold and colleagues.531 These authors disagreed, however, about the ability of endovaginal ultrasound to detect focal adenomyosis, with the former and the latter groups reporting 53% and 89%, respectively.

Endometriosis and Uterine Contractility Adenomyosis is likely associated with myometrial dysfunction,535,536 possibly associated with endometriosis-types of alterations of the eutopic endometrium537 and the JZ.27 In a provocative series of publications, Leyendecker’s team promoted the concept that endometriosis is associated with a hyperkinetic-dyskinetic condition of the uterus473,538 as a result of structural abnormalities of the uterine wall,539

CHAPTER 35  Pelvic Imaging in Reproductive Endocrinology

which impairs the retrograde transport of sperm during the follicular phase,24,231,540 and the proper antegrade emptying of uterine content at the time of menses.236 From their studies using the uterine displacement of 99Tc-labeled MAA, these authors observed two pathological findings in endometriosis: (1) a pathological increase in retrograde transport at the time of menses; and (2) the loss of retrograde transport targeted toward the tube that faces the developing follicle during the late follicular phase of natural cycles.24,235 Both phenomena could contribute to the infertility that accompanies even the milder forms of endometriosis. The original report of activation of the aromatase gene in the eutopic endometrium in case of endometriosis541 that has been amply confirmed542 offers a possible explanation for the local state of hyperestrogenism purported to be the cause for the relative progesterone resistance542,543 and the dyskinetic alterations encountered in endometriosis.241,544 That COH-IUI cycles have markedly lower pregnancy rates in endometriosis as compared to unaffected controls545-547 is consistent with the concept that endometriosis is accompanied by a sperm transport problem stemming from uterine dyskinesia. The complete reference list can be found on the companion Expert Consult Web site at www.expertconsult.com.

Suggested Readings Allison SJ, et al: Saline-infused sonohysterography: tips for achieving greater success, Radiographics 31:1991–2004, 2011. Benagiano G, et al: The pathophysiology of uterine adenomyosis: an update, Fertil Steril 98:572–579, 2012. Chapron C, et  al: Magnetic resonance imaging and endometriosis: deeply infiltrating endometriosis does not originate from the rectovaginal septum, Gynecol Obstet Invest 53:204–208, 2002. Chapron C, et al: Presurgical diagnosis of posterior deep infiltrating endometriosis based on a standardized questionnaire, Hum Reprod 20: 507–513, 2005. de Kroon CD, et al: Saline contrast hysterosonography in abnormal uterine bleeding: a systematic review and meta-analysis, BJOG 110:938–947, 2003.

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Deb S, et al: Quantitative analysis of antral follicle number and size: a comparison of two-dimensional and automated three-dimensional ultrasound techniques, Ultrasound Obstet Gynecol 35:354–360, 2010. Fanchin R, et al: New look at endometrial echogenicity: objective computerassisted measurements predict endometrial receptivity in in vitro fertilization-embryo transfer, Fertil Steril 74:274–281, 2000. Hamilton JA, et al: Routine use of saline hysterosonography in 500 consecutive, unselected, infertile women, Hum Reprod 13:2463–2473, 1998. Jayaprakasan K, et al: Prediction of in vitro fertilization outcome at different antral follicle count thresholds in a prospective cohort of 1,012 women, Fertil Steril 98:657–663, 2012. Kaufman RH, et al: Upper genital tract changes associated with exposure in utero to diethylstilbestrol, Am J Obstet Gynecol 128:51–59, 1977. Kunz G, et  al: Sonographic evidence for the involvement of the uteroovarian counter-current system in the ovarian control of directed uterine sperm transport, Hum Reprod Update 4:667–672, 1998. La Marca A, et al: Anti-mullerian hormone (AMH) as a predictive marker in assisted reproductive technology (ART), Hum Reprod Update 16: 113–130, 2010. Leyendecker G, et  al: Endometriosis: a dysfunction and disease of the archimetra, Hum Reprod Update 4:752–762, 1998. Lindheim SR, et  al: Ultrasound guided embryo transfer significantly improves pregnancy rates in women undergoing oocyte donation, Int J Gynaecol Obstet 66:281–284, 1999. Maheshwari A, et al: Adenomyosis and subfertility: a systematic review of prevalence, diagnosis, treatment and fertility outcomes, Hum Reprod Update 18:374–392, 2012. Ng EH, et  al: Comparison of 2-dimensional, 3-dimensional, and vascular ultrasonographic parameters for endometrial receptivity between 2 consecutive stimulated in  vitro fertilization cycles, J Ultrasound Med 26:931–939, 2007. Saba L, et  al: MRI and “tenderness guided” transvaginal ultrasonography in the diagnosis of recto-sigmoid endometriosis, J Magn Reson Imaging 35:352–360, 2012. Saccardi C, et  al: Comparison between transvaginal ultrasound, sonovaginography and magnetic resonance imaging in the diagnosis of posterior deep infiltrating endometriosis, Ultrasound Obstet Gynecol, Jan 17 2012. [Epub ahead of print]. Tur-Kaspa I, et  al: A prospective evaluation of uterine abnormalities by saline infusion sonohysterography in 1,009 women with infertility or abnormal uterine bleeding, Fertil Steril 86:1731–1735, 2006. Vodolazkaia A, et  al: Evaluation of a panel of 28 biomarkers for the noninvasive diagnosis of endometriosis, Hum Reprod 27:2698–2711, 2012.

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