New technique for automatic sonographic measurement of change in head-perineum distance and angle of progression during active phase of second stage of labor.

OBJECTIVE: To evaluate the performance of a new ultrasound technique for the automatic assessment of the change in head-perineum distance (delta-HPD) and angle of progression (delta-AoP) during the active phase of the second stage of labor. METHODS: This was a prospective observational cohort study including singleton term pregnancies with fetuses in cephalic presentation during the active phase of the second stage of labor. In each patient, two videoclips of 10 s each were acquired transperineally, one in the axial and one in the sagittal plane, between rest and the acme of an expulsive effort, in order to measure HPD and AoP, respectively. The videoclips were processed offline and the difference between the acme of the pushing effort and rest in HPD (delta-HPD) and AoP (delta-AoP) was calculated, first manually by an experienced sonographer and then using a new automatic technique. The reliability of the automatic algorithm was evaluated by comparing the automatic measurements with those obtained manually, which was considered as the reference gold standard. RESULTS: Overall, 27 women were included. A significant correlation was observed between the measurements obtained by the automatic and the manual methods for both delta-HPD (intraclass correlation coefficient (ICC) = 0.97) and delta-AoP (ICC = 0.99). The high accuracy provided by the automatic algorithm was confirmed by the high values of the coefficient of determination (r2 = 0.98 for both delta-HPD and delta-AoP) and the low residual errors (root mean square error = 1.2 mm for delta-HPD and 1.5° for delta-AoP). A Bland-Altman analysis showed a mean difference of 0.52 mm (limits of agreement, -1.58 to 2.62 mm) for delta-HPD (P = 0.034) and 0.35° (limits of agreement, -2.54 to 3.09°) for delta-AoP (P = 0.39) between the manual and automatic measurements. CONCLUSIONS: The automatic assessment of delta-AoP and delta-HPD during maternal pushing efforts is feasible. The automatic measurement of delta-AoP appears to be reliable when compared with the gold standard manual measurement by an experienced operator. Copyright © 2020 ISUOG. Published by John Wiley & Sons Ltd.

Developmental mutant mouse models for external genitalia formation.

Development of external genitalia and perineum is the subject of developmental biology as well as toxicology and teratology researches. Cloaca forms in the lower (caudal) end of endoderm. Such endodermal epithelia and surrounding mesenchyme interact with various signals to form the external genitalia. External genitalia (the anlage termed as genital tubercle: GT) formation shows prominent sexually dimorphic morphogenesis in late embryonic stages, which is an unexplored developmental research field because of many reasons. External genitalia develop adjacent to the cloaca which develops urethra and corporal bodies. Developmental regulators including growth factor signals are necessary for epithelia-mesenchyme interaction (EMI) in posterior embryos including the cloaca and urethra in the genitalia. In the case of male type urethra, formation of tubular urethra proceeds from the lower (ventral) side of external genitalia as a masculinization process in contrast to the case of female urethra. Mechanisms for its development are not elucidated yet due to the lack of suitable mutant mouse models. Because of the recent progresses of Cre (recombinase)-mediated conditional target gene modification analyses, many developmental regulatory genes become increasingly analyzed. Conditional gene knockout mouse approaches and tissue lineage approaches are expected to offer vital information for such sexually dimorphic developmental processes. This review aims to offer recent updates on the progresses of these emerging developmental processes for the research field of congenital anomalies.

Anogenital distance plasticity in adulthood: implications for its use as a biomarker of fetal androgen action.

Androgen action during the fetal masculinization programming window (MPW) determines the maximum potential for growth of androgen-dependent organs (eg, seminal vesicles, prostate, penis, and perineum) and is reflected in anogenital distance (AGD). As such, determining AGD in postnatal life has potential as a lifelong easily accessible biomarker of overall androgen action during the MPW. However, whether the perineum remains androgen responsive in adulthood and thus responds plastically to perturbed androgen drive remains unexplored. To determine this, we treated adult male rats with either the antiandrogen flutamide or the estrogen diethylstilbestrol (DES) for 5 weeks, followed by a 4-week washout period of no treatment. We determined AGD and its correlate anogenital index (AGI) (AGD relative to body weight) at weekly intervals across this period and compared these with normal adult rats (male and female), castrated male rats, and appropriate vehicle controls. These data showed that, in addition to reducing circulating testosterone and seminal vesicle weight, castration significantly reduced AGD (by ∼17%), demonstrating that there is a degree of plasticity in AGD in adulthood. Flutamide treatment increased circulating testosterone yet also reduced seminal vesicle weight due to local antagonism of androgen receptor. Despite this suppression, surprisingly, flutamide treatment had no effect on AGD at any time point. In contrast, although DES treatment suppressed circulating testosterone and reduced seminal vesicle weight, it also induced a significant reduction in AGD (by ∼11%), which returned to normal 1 week after cessation of DES treatment. We conclude that AGD in adult rats exhibits a degree of plasticity, which may be mediated by modulation of local androgen/estrogen action. The implications of these findings regarding the use of AGD as a lifelong clinical biomarker of fetal androgen action are discussed.

Nonmyocytic androgen receptor regulates the sexually dimorphic development of the embryonic bulbocavernosus muscle.

The bulbocavernosus (BC) is a sexually dimorphic muscle observed only in males. Androgen receptor knockout mouse studies show the loss of BC formation. This suggests that androgen signaling plays a vital role in its development. Androgen has been known to induce muscle hypertrophy through satellite cell activation and myonuclei accretion during muscle regeneration and growth. Whether the same mechanism is present during embryonic development is not yet elucidated. To identify the mechanism of sexual dimorphism during BC development, the timing of morphological differences was first established. It was revealed that the BC was morphologically different between male and female mice at embryonic day (E) 16.5. Differences in the myogenic process were detected at E15.5. The male BC possesses a higher number of proliferating undifferentiated myoblasts. To identify the role of androgen signaling in this process, muscle-specific androgen receptor (AR) mutation was introduced, which resulted in no observable phenotypes. Hence, the expression of AR in the BC was examined and found that the AR did not colocalize with any muscle markers such as Myogenic differentiation 1, Myogenin, and paired box transcription factor 7. It was revealed that the mesenchyme surrounding the BC expressed AR and the BC started to express AR at E15.5. AR mutation on the nonmyocytic cells using spalt-like transcription factor 1 (Sall1) Cre driver mouse was performed, which resulted in defective BC formation. It was revealed that the number of proliferating undifferentiated myoblasts was reduced in the Sall1 Cre:AR(L-/Y) mutant embryos, and the adult mutants were devoid of BC. The transition of myoblasts from proliferation to differentiation is mediated by cyclin-dependent kinase inhibitors. An increased expression of p21 was observed in the BC myoblast of the Sall1 Cre:AR(L-/Y) mutant and wild-type female. Altogether this study suggests that the nonmyocytic AR may paracrinely regulate the proliferation of myoblast possibly through inhibiting p21 expression in myoblasts of the BC.

Embryonic origin and remodeling of the urinary and digestive outlets.

Separating digestive and urinary outlets is a critical step during mammalian embryogenesis. However, the natural history of these structures is poorly studied, and little is known about their embryonic origin. Here, we show that peri-cloacal mesenchymal (PCM) progenitors are the major source of these structures. Surprisingly, PCM progenitors also contribute to perineum, a structural barrier separating the urinary and digestive tracts, suggesting a potential role of PCM progenitors in establishing independent urinary and digestive outlets. We demonstrate that Six1 and Six2 are complementarily but asymmetrically expressed in the PCM progenitors. Deletion of these genes results in decreased cell survival and proliferation, and consequently in agenesis of the perineum and severe hypoplasia of the genital tubercle. Together, these findings suggest that PCM progenitors are the unexpected source of perineum and genital tubercle, and establish a basic framework for investigating normal and abnormal development of anorectal and genitourinary structures.

Clear cells of Toker in the developing anogenital region of male and female fetuses.

The clear cells of Toker are a mysterious population of intra-epidermal glandular cells. They were originally described in nipples, but were recently observed in the vulva as well. It was hypothesized that intra-epidermal embryonic remnants or underlying glands were a potential source. The embryological aspects were investigated by studying specimens of the anogenital region of 18 male and 15 female fetuses between 12 and 39 weeks gestation. The search for Toker cells was enhanced by cytokeratin (CK) 7 immunohistochemistry. The investigation showed that Toker cell elements are a normal, though highly variable constituent of the developing anogenital region. The study revealed the following: (1) single intra-epidermal glandular vesicles near follicular anlages in interlabial sulcuses of female fetuses of 15 and 16.5 weeks gestation; (2) CK7+ solitary cells, clusters, and vesicles which were related to developing intra-epidermal follicular canal tracks and tended to disperse inside the epidermis in fetuses of approximately 18 weeks gestation; (3) dispersed CK7+ cells in fetuses of 19-23 weeks gestation; (4) characteristic CK7+ Toker cell proliferations in fetuses more than 23 weeks gestation. These observations indicate that in the anogenital region, primordial follicular cells programmed to participate in the formation of apocrine and mammary-like glands, become displaced into the epidermis where they disperse, and proliferate into Toker cell populations. However, the proximity of Toker cells to CK7+ cells in excretory ducts of late fetal apocrine and mammary-like glands suggested a possible additional source. Consequences for Toker cells of the breast and primary Paget disease are discussed.

False positive reactivity of a substance P-antibody in the ectodermal/epithelial plug of the nose, ear, eye and perineum of the human and mouse fetuses.

Epithelial/ectodermal plug formation in the developing nose, ear, and eye regions is followed by canalization/recanalization mediated by cell death. However, the mechanism is not well understood. Recently, substance P (SP)-mediated cell death, rather than cell apoptosis, has been reported in neuronal and non-neuronal cells. Horizontal paraffin sections of 5 human fetuses at 15-16 weeks of gestation were used to examine the entire area of the nose, ear, eye and perineum with immunohistochemistry for SP and its receptor neurokinin-1 (NK-1), and protein gene product (PGP) 9.5 and S100 protein to identify whether the positive cells had neural origins. The deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method was also conducted to identify apoptosis. Four SP antibodies were commercially obtained and compared the results. In addition, using the same antibodies for SP, those results were compared with fetal mouse heads (E14-17). Substance P immunoreactivity of one of the 4 antibodies (sc9758) was clearly found in the nasal plug, the epithelium of the anterior nasal cavity, the entire excretory tear duct, the marginal palpebral conjunctiva, the auditory meatal plug, the parotid duct, the external urethral orifice and, the preputial lamella along the future prepuce. Immunoreactivity was usually seen in enlarged round cells in humans. In fetal mouse heads, in spite of negative reaction in all these sites, the midline epithelial seam at the palate fusion and the oral epithelium especially at and near the tooth germ specifically reacted with the sc9758. Nevertheless, the other 3 antibodies did not react at any of those sites both in human and mouse fetuses. NK-1 receptor-positive cells were seen in the nose and meatal plugs and preputial lamella, but not in the tear duct. S100 protein, PGP 9.5, and TUNEL method all demonstrated negative reactivity at any sc9758-positive sites. Consequently, the present immunoreactivity of the sc9758 antibody seemed to be false positive, but it was likely to react with a specific substance in the epithelial or ectodermal cell because of the clearly restricted staining. Which substance it crossed remains unknown.

Development of the external anal sphincter with special reference to intergender difference: observations of mid-term fetuses (15-30 weeks of gestation).

To investigate intergender differences in muscle cleavage and joining during development of the external anal sphincter (EAS), we examined semiserial sections of 16 fetuses between 15 and 30 weeks of gestation (6 males and 10 females). The subcutaneous part of the EAS (EASsc) developed along the male perineal raphe and extended posteriorly. Thus, the male EAS was characterized by anterior protrusion of the subcutaneous muscle, in contrast to the almost circular female EAS. In both genders, the bulbospongiosus anlage (or the levator ani anlage) issued muscle fibers to form the superficial (or deep) part of the EAS. The EASsc communicated with the superficial part in males, whereas the female bulbospongiosus tended to communicate with the levator ani rather than the EAS. In both genders, the longitudinal muscle bundle(s) of the anorectum contributed to perineal body formation. However, the male perineal body also had a thick fascia between the rhabdosphincter and the levator. The bulbospongiosus seems to play a critical role in forming the EAS. A strict intergender difference in subcutaneous muscle development is evident along the perineal raphe, as the raphe is not evident in females. These results help to explain variations in the EAS, including anal malformations.

Developmental instability of ano-genital distance index: Implications for assessment of prenatal masculinization.

Prenatal androgens are generally assumed to permanently affect the morphology of external genitalia. In laboratory rodents, the ano-genital distance index (AGDI) has become widely used as a marker of natural prenatal masculinization. A greater value should indicate a greater masculinization. The aim of this study was to assess the developmental stability of the AGDI in female mice during pre- (Days 1-21) and postweaning (Days 21-61) periods. Presuming that the AGDI reflects a permanent effect of prenatal masculinization, we hypothesized that the AGDI will be a developmentally stable morphometric measurement (interindividual differences in the AGDI measurement should persist over time). In contrast to our prediction, the AGDI showed poor repeatability both during pre- (.15) and postweaning (.22) periods, indicating developmental instability. (A value of 1 indicates perfect repeatability and a value of 0 indicates no repeatability.) The AGDI thus does not seem to be a reliable marker of prenatal female masculinization.

Ruptured rectal duplication cyst with classical bladder exstrophy.

A newborn boy was brought to us, 2 hours after birth, with a mucosal-lined left hemiperineal lesion associated with classical bladder exstrophy and an anterolaterally displaced anus. Perineal anatomy was restored by excising the mucosa lined lesion. The bladder closure for classical bladder exstrophy was done at the same time. Histologically, gastric, respiratory, and small intestinal epithelia were present in the mucosa. A rectal duplication cyst that had ruptured in utero through the hemiperineum could explain the anomaly. The association of classical bladder exstrophy with ruptured rectal duplication cyst has never previously been described in the literature.

Ultrasound assessment of fetal head-perineum distance before induction of labor.

OBJECTIVES: To evaluate fetal head-perineum distance measured by ultrasound imaging as a predictive factor for induction of labor, and to compare this distance with maternal factors, the Bishop score and ultrasound measurements of cervical length, cervical angle and occiput position. METHODS: The study included 275 women admitted for induction of labor. The fetal head-perineum distance was measured by transperineal ultrasound imaging as the shortest distance from the outer bony limit of the fetal skull to the skin surface of the perineum. Cervical length and angle was measured by transvaginal ultrasound examination, and fetal head position was assessed by transabdominal ultrasound imaging. The Bishop score was assessed without knowledge of ultrasound measurements. Receiver-operating characteristics (ROC) curves were used for evaluation of the probability of a successful vaginal delivery. The time from induction to delivery was tested using Cox regression analysis with ultrasound measurements, parity and body mass index (BMI) as possible predictive factors. RESULTS: Areas under the ROC curve for prediction of vaginal delivery were 62% (95% CI, 52-71%) for fetal head-perineum distance (P = 0.03), 61% (95% CI, 51-71%) for cervical length (P = 0.03), 63% (95% CI, 52-74%) for cervical angle (P = 0.02), 61% (95% CI, 52-70%) for Bishop score (P = 0.03) and 60% (95% CI, 51-69%) for BMI (P = 0.05). The Cesarean delivery rate was 22% among nulliparous and 5% among parous women (P < 0.01). Parity, fetal head-perineum distance, cervical length and cervical angle were contributing factors predicting vaginal delivery within 24 h in a Cox regression model. Occiput posterior position had no significant predictive value. CONCLUSIONS: Fetal head-perineum distance measured by transperineal ultrasound examination can predict vaginal delivery after induction of labor, with a predictive value similar to that of ultrasonographically measured cervical length and the Bishop score. However, we judge none of these methods used alone to be good enough in a clinical setting.

Cell lineage analysis demonstrates an endodermal origin of the distal urethra and perineum.

Congenital malformations of anorectal and genitourinary (collectively, anogenital) organs occur at a high frequency in humans, however the lineage of cells that gives rise to anogenital organs remains poorly understood. The penile urethra has been reported to develop from two cell populations, with the proximal urethra developing from endoderm and the distal urethra forming from an apical ectodermal invagination, however this has never been tested by direct analysis of cell lineage. During gut development, endodermal cells express Sonic hedgehog (Shh), which is required for normal patterning of digestive and genitourinary organs. We have taken advantage of the properties of Shh expression to genetically label and follow the fate of posterior gut endoderm during anogenital development. We report that the entire urethra, including the distal (glandar) region, is derived from endoderm. Cloacal endoderm also gives rise to the epithelial linings of the bladder, rectum and anterior region of the anus. Surprisingly, the lineage map also revealed an endodermal origin of the perineum, which is the first demonstration that endoderm differentiates into skin. In addition, we fate mapped genital tubercle ectoderm and show that it makes no detectable contribution to the urethra. In males, formation of the urethral tube involves septation of the urethral plate by continued growth of the urorectal septum. Analysis of cell lineage following disruption of androgen signaling revealed that the urethral plate of flutamide-treated males does not undergo this septation event. Instead, urethral plate cells persist to the ventral margin of the tubercle, mimicking the pattern seen in females. Based on these spatial and temporal fate maps, we present a new model for anogenital development and suggest that disruptions at specific developmental time points can account for the association between anorectal and genitourinary defects.

The development of the male genitourinary system. I. The origin of the urorectal septum and the formation of the perineum.

The embryological development of the male urinary system remains a subject of much controversy. As a result the pathogenesis of congenital anomalies such as hypospadias and epispadias, which are presented to the reconstructive surgeon remains poorly understood. A review of the literature identifies its three principal developmental stages: (1) division of the cloaca into the urogenital sinus and hindgut by the urorectal septum and the formation of the perineum; (2) the extension of the cloaca and its epithelium in the form of the urethral plate through the developing genital tubercle; (3) the separation of this extension from the surface during the formation of the urethra. This study, which uses a mouse model, examines these developmental stages in detail and together with a comprehensive review of the literature resolves many of the controversies relating to the development of the male urinary system. It reveals new insights into the origin of the associated congenital defects.

Neuroanatomy of the male urethra and perineum.

OBJECTIVE: To describe the topography of the perineal nerves from their pudendal origin to their course into the male genitalia, with specific attention on the course of the perineal nerve along the ventral penis, including branches into bulbospongiosus muscle and corpus spongiosum. MATERIALS AND METHODS: The study comprised 18 normal human fetal penile specimens at 17.5-38 weeks of gestation (determined by fetal heel-to-toe length). Specimens were fixed in formalin, embedded in paraffin wax and serially sectioned at 6 micro m. The penile specimens contained the whole penis from the glans to the crural bodies, beneath the pubic arch and the perineum up to the anal verge. Immunocytochemistry was assessed on selected sections with antibodies against the neuronal markers S-100 and nitric oxide synthase (nNOS). Three-dimensional computer reconstruction of serial sections allowed an in-depth analysis of the neuroanatomy of the fetal penis, perineum and surrounding structures. RESULTS: After the pudendal nerve leaves the pudendal canal it gives rise to the perineal nerve branches in the ischiorectal fossa. Perineal nerves travel alongside the ischiocavernous and bulbospongiosus muscles and before reaching the latter, nerve branches course into the bulbospongiosus muscle. During its pathway within this muscle, fine nerve fibres course into the corpus spongiosum by piercing through the junction of the muscle. At the penoscrotal area, the perineal nerves give branches to the scrotum, funnelling into the interscrotal septum. Perineal nerves continue their pathway over the ventral side of penis covering the ventral surface of corpus spongiosum. Branches of the dorsal nerve of the penis at the junction of corpus cavernosum and corpus spongiosum assemble into a network with the perineal nerves. All perineal nerves from their main trunk at the ischiorectal fossa until their interaction with dorsal nerve of penis at the base of penis were nNOS negative. After the interaction with the dorsal nerve of penis, they become nNOS positive. CONCLUSION: Integrating neuroanatomical knowledge about the perineal nerves and their communication with the dorsal nerve of penis should facilitate a strategic approach to reconstructive procedures on the penis. Special care should be taken at the junction between the corpora cavernosa and spongiosa, where the dorsal nerve joins the perineal nerve, and at the proximal bulbospongiosus muscle, thereby protecting the fine nerves piercing into the cavernosa spongiosa.

Sexual dimorphism of perineal muscles and motoneurons in spotted hyenas.

Female spotted hyenas are known for their male-like genitalia, high levels of aggression, and dominance over males, characteristics which are attributed to exposure to elevated levels of testosterone in utero. Although the nervous system of spotted hyenas has not previously been examined, one might predict that neural systems which are sexually dimorphic in other mammals would be monomorphic in this species. Spinal motoneurons which innervate muscles associated with the phallus are located in Onuf's nucleus and are more numerous in males than in females in a wide array of mammals. Onuf's nucleus was examined in adult and neonatal spotted hyenas and, contrary to expectation, was found to be sexually dimorphic in the typical mammalian pattern: Males have significantly more motoneurons in Onuf's nucleus than do females. This dimorphism was correlated with a previously undescribed dimorphism in the relevant target musculature. Specifically, the morphology of the bulbocavernosus muscle is distinctly different in male and female spotted hyenas. Pregnant hyenas were treated with anti-androgen in an attempt to interfere with the actions of androgen during fetal development. Motoneuron number in Onuf's nucleus and the morphology of the bulbocavernosus muscle were feminized in males exposed to anti-androgen in utero.

Ano vulvar congénito / Congenital vulvar anus

Presentamos un trabajo sobre ano vulvar congénito, en una paciente de 19 años de edad. Por lo infrecuente de la lesión, mas por tratarse de un adulto, hemos querido hacer un esbozo de las diversas malformaciones congénitas del periné y la embriología del mismo. Detalladamente se describe la técnica quirúrgica utilizada para la corrección de esta anomalía y la evolución postoperatoria de la paciente

Fetal anoperineogenital images by ultrasonography--regarding the breech and transverse presentations.

Reports seeking the rate of successful echography of the fetal scrotum, penis and labia majora classified by individual weeks of gestation over the long period from the 22nd to the 41st week of gestation, are few. In the present study, the rates of successful echography of the scrotum, penis, labia majora, M-type and F-type of the fetal APG region in the breech and transverse presentations in each week of gestation for the period from the 22nd to the 41st week of gestation were pursued with a view to finding out the best suited time for the observation of each image. Ultrasonography was performed 3039 times in total on 620 pregnant women who delivered their babies, and the following results were obtained: The rates of successful echography classified by presentation decreased in the order of cephalic, breech and transverse presentations. The rates of successful echography with the first and second manipulations classified by individual weeks of gestation were also determined, and the optimal periods for observation were sought. The rates of correct echography as a whole were also determined, with the following results: Breech presentation is 74.1% in male fetuses and 68.3% in female fetuses; Transverse presentation is 66.2% in male fetuses and 58.6% in female fetuses, respectively.