Evaluation of ambiguous genitalia.

PURPOSE OF REVIEW: To provide a framework for the evaluation of ambiguous genitalia. RECENT FINDINGS: The most pressing evaluation of ambiguous genitalia is assessment for life-threatening causes such as salt-wasting congenital adrenal hyperplasia (CAH) or syndromes with underlying anomalies such as neurologic or cardiac malformations. A multidisciplinary team, including specialists in Gynecology, Endocrinology, Urology, Genetics, Clinical Psychology/Psychiatry, Radiology, Nursing, Neonatology, and Pediatric Surgery, should be involved. Each patient should be approached in an individualized manner to assign sex of rearing in the most expeditious yet thoughtful means possible.As knowledge on the natural history of sex preference and fertility of individuals with ambiguous genitalia increases, controversy regarding the indication for and timing of genital surgery continues. Considerations include gender identity, future fertility, malignancy risk, infection prevention, and functional anatomy for sexual activity. SUMMARY: The evaluation of ambiguous genitalia should involve a multidisciplinary team. A combination of history taking, physical examination, laboratory evaluation, and radiologic assessment can assist with the diagnosis. Care should be taken to emphasize karyotypic sex is not equivalent to gender and to use caution with regards to irreversible medical and surgical therapies which may impact fertility and sexual function and nonconform with future sex identity.

Expression and ontogeny of growth hormone (Gh) in the protogynous hermaphroditic ricefield eel (Monopterus albus).

Growth hormone (GH) is a single-chain polypeptide hormone mainly secreted by somatotropes of the anterior pituitary gland and is an important regulator of somatic growth in vertebrates including teleosts. In this study, a polyclonal antiserum against ricefield eel Gh was generated and the expression of Gh at the mRNA and protein levels was analyzed. Both RT-PCR and western blot analysis showed that Gh was predominantly expressed in the pituitary glands of ricefield eels. The immunoreactive Gh signals were localized to the multicellular layers of the adenohypophysis adjacent to the neurohypophysis in ricefield eels. Ontogenetic analysis showed that immunoreactive Gh signals could be detected in the pituitary glands of ricefield eel embryos as early as 3 days post-fertilization. During the sex change from female to male, the levels of the immunoreactive Gh signals in the pituitary glands of the ricefield eels peaked at the intersexual stage. These results suggest that Gh in the pituitary glands may be associated with embryonic development before hatching, as well as with the sex change in the adult ricefield eels, possibly via the classical endocrine manner.

Intersex gonad differentiation in cultured Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeon.

Among sturgeons, the occurrence of individuals with gonads containing both testis and ovary components is considered pathological, and such fish are described as intersex individuals or intersexes. Intersexes are observed in both wild and cultured populations of sturgeon, usually at low frequencies. In the present study, intersex Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeons constituted 30% of the studied populations. Macroscopically, intersex gonads were recognizable from 500 days posthatching (dph). Initially, gonads with predominantly male characteristics (testis-ova) were observed, but in older fish gonads with predominantly female traits (ova-testis) were more frequent. Using microscopic analysis, intersex gonads were discernible by 130-200 dph. Observations of intersex germinal epithelium development and analysis of sex distribution in the study populations indicated that feminization was occurring. Histological analysis revealed that differentiation of the germinal epithelium in such gonads was accompanied by various morphological alterations (transformations) that were described using quantitative and localization criteria. The most common type of transformations, massive subepithelial transformations, was manifested by the presence of abundant female germinal tissue located under the gonad surface epithelium in the developing testis. These transformations were identified in the early development stage (100-200 dph). In this type of transformation, differentiation of female germinal tissue at the gonad surface and male tissue at the mesorchium/mesovarium resulted in complete formation of both male and female germinal epithelia within the same gonad.

Bifid scrotum and anocutaneous fistula associated with a perineal lipomatous tumor complicated by temporary bilateral cryptorchidism in utero mimicking ambiguous genitalia: 2-D/3-D fetal ultrasonography.

Ambiguous genitalia (AG) is a morphological diagnosis defined as genitalia not typical of a male or female. Findings mimicking AG, such as penoscrotal anomalies, anorectal malformations, and perineal lipomatous tumors, may prevent accurate identification of the fetal sex. We report a case of bifid scrotum and anocutaneous fistula associated with a perineal lipomatous tumor complicated by temporary bilateral cryptorchidism in utero, which were findings mimicking AG. Several perineal anomalies are associated developmental occurrences. In the present case, the combination of bifid scrotum and temporary bilateral cryptorchidism in the male fetus mimicked the combination of clitoromegaly and prominent labia, which are commonly observed in female fetuses. However, serial systemic assessments using prenatal 2-D/3-D ultrasonography and magnetic resonance imaging were unable to detect the anocutaneous fistula and differentiate the perineal lipomatous tumor. This case report suggests that the prenatal detection of perineal abnormalities may warn obstetricians of potentially undetected congenital perineal anomalies.

Male pseudohermaphroditism caused by mutations of testicular 17 beta-hydroxysteroid dehydrogenase 3.

Defects in the conversion of androstenedione to testosterone in the fetal testes by the enzyme 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) give rise to genetic males with female external genitalia. We have used expression cloning to isolate cDNAs encoding a microsomal 17 beta-HSD type 3 isozyme that shares 23% sequence identity with other 17 beta-HSD enzymes, uses NADPh as a cofactor, and is expressed predominantly in the testes. The 17 beta HSD3 gene on chromosome 9q22 contains 11 exons. Four substitution and two splice junction mutations were identified in the 17 beta HSD3 genes of five unrelated male pseudohermaphrodites. The substitution mutations severely compromised the activity of the 17 beta-HSD type 3 isozyme.

Sox10 gain-of-function causes XX sex reversal in mice: implications for human 22q-linked disorders of sex development.

Male development in mammals is normally initiated by the Y-linked gene Sry, which activates expression of Sox9, leading to a cascade of gene activity required for testis formation. Although defects in this genetic cascade lead to human disorders of sex development (DSD), only a dozen DSD genes have been identified, and causes of 46,XX DSD (XX maleness) other than SRY translocation are almost completely unknown. Here, we show that transgenic expression of Sox10, a close relative of Sox9, in gonads of XX mice resulted in development of testes and male physiology. The degree of sex reversal correlated with levels of Sox10 expression in different transgenic lines. Sox10 was expressed at low levels in primordial gonads of both sexes during normal mouse development, becoming male-specific during testis differentiation. SOX10 protein was able to activate transcriptional targets of SOX9, explaining at a mechanistic level its ability to direct male development. Because over-expression of SOX10 alone is able to mimic the XX DSD phenotypes associated with duplication of human chromosome 22q13, and given that human SOX10 maps to 22q13.1, our results functionally implicate SOX10 in the etiology of these DSDs.

Masculine epigenetic sex marks of the CYP19A1/aromatase promoter in genetically male chicken embryonic gonads are resistant to estrogen-induced phenotypic sex conversion.

Sex of birds is genetically determined through inheritance of the ZW sex chromosomes (ZZ males and ZW females). Although the mechanisms of avian sex determination remains unknown, the genetic sex is experimentally reversible by in ovo exposure to exogenous estrogens (ZZ-male feminization) or aromatase inhibitors (ZW-female masculinization). Expression of various testis- and ovary-specific marker genes during the normal and reversed gonadal sex differentiation in chicken embryos has been extensively studied, but the roles of sex-specific epigenetic marks in sex differentiation are unknown. In this study, we show that a 170-nt region in the promoter of CYP19A1/aromatase, a key gene required for ovarian estrogen biosynthesis and feminization of chicken embryonic gonads, contains highly quantitative, nucleotide base-level epigenetic marks that reflect phenotypic gonadal sex differentiation. We developed a protocol to feminize ZZ-male chicken embryonic gonads in a highly quantitative manner by direct injection of emulsified ethynylestradiol into yolk at various developmental stages. Taking advantage of this experimental sex reversal model, we show that the epigenetic sex marks in the CYP19A1/aromatase promoter involving DNA methylation and histone lysine methylation are feminized significantly but only partially in sex-converted gonads even when morphological and transcriptional marks of sex differentiation show complete feminization, being indistinguishable from gonads of normal ZW females. Our study suggests that the epigenetic sex of chicken embryonic gonads is more stable than the morphologically or transcriptionally characterized sex differentiation, suggesting the importance of the nucleotide base-level epigenetic sex in gonadal sex differentiation.

Symmetrical division of mouse oocytes during meiotic maturation can lead to the development of twin embryos that amalgamate to form a chimeric hermaphrodite.

BACKGROUND: Gentle compression of mouse oocytes during meiosis-1 prevented the usual extrusion of a small polar body and resulted in the symmetrical division of the ooplasm into two cells of similar size within the zona pellucida. The purpose of our study was to determine whether such cells, equivalent to two small oocytes, were capable of embryonic development and would result in birth following transfer to the uterus. METHODS: IVF of the 2-celled oocytes was performed and the twin intra-zonal embryos were observed. In each case, the two embryos that originated from fertilized cells with two pronuclei were observed to amalgamate and form a single morula and subsequent blastocyst that was transferred to the uterus of a recipient of a different mouse strain. FISH analysis was performed on sectioned paraffin-embedded tissue of the offspring. RESULTS: In symmetrically divided oocytes each cell contained a metaphase II spindle. Both cells were fertilizable and cleaved to form twin embryos within the same zona pellucida. Most twin embryos amalgamated to form a single compacted morula, which progressed to hatched blastocysts that contained a single inner cell mass. In total, 104 of these blastocysts were transferred to 19 mice, two of which became pregnant, resulting in the birth of three offspring. FISH analysis showed that one newborn contained both XX and XY cells. CONCLUSIONS: We found that two small oocytes fertilized within the same zona pellucida to form twin embryos that amalgamate to establish a single chimeric embryo. This may be one mechanism that leads to the formation of a chimeric hermaphrodite when an embryo containing XX cells mixes with its intra-zonal twin containing XY cells.

Generation of hermaphrodite transgenic papaya lines with virus resistance via transformation of somatic embryos derived from adventitious roots of in vitro shoots.

Papaya production is seriously limited by Papaya ringspot virus (PRSV) worldwide and Papaya leaf-distortion mosaic virus (PLDMV) in Eastern Asia. An efficient transformation method for developing papaya lines with transgenic resistance to these viruses and commercially desirable traits, such as hermaphroditism, is crucial to shorten the breeding program for this fruit crop. In this investigation, an untranslatable chimeric construct pYP08 containing truncated PRSV coat protein (CP) and PLDMV CP genes coupled with the 3' untranslational region of PLDMV, was generated. Root segments from different portions of adventitious roots of in vitro multiple shoots of hermaphroditic plants of papaya cultivars 'Tainung No. 2', 'Sunrise', and 'Thailand' were cultured on induction medium for regeneration into somatic embryos. The highest frequency of somatic embryogenesis was from the root-tip segments of adventitious roots developed 2-4 weeks after rooting in perlite medium. After proliferation, embryogenic tissues derived from somatic embryos were wounded in liquid-phase by carborundum and transformed by Agrobacterium carrying pYP08. Similarly, another construct pBG-PLDMVstop containing untranslatable CP gene of PLDMV was also transferred to 'Sunrise' and 'Thailand', the parental cultivars of 'Tainung No. 2'. Among 107 transgenic lines regenerated from 349 root-tip segments, nine lines of Tainung No. 2 carrying YP08 were highly resistant to PRSV and PLDMV, and 9 lines (8 'Sunrise' and 1 'Thailand') carrying PLDMV CP highly resistant to PLDMV, by a mechanism of post-transcriptional gene silencing. The hermaphroditic characteristics of the transgenic lines were confirmed by PCR with sex-linked primers and phenotypes of flower and fruit. Our approach has generated transgenic resistance to both PRSV and PLDMV with commercially desirable characters and can significantly shorten the time-consuming breeding programs for the generation of elite cultivars of papaya hybrids.

Embryology and disorders of sexual development.

Recent consensus is that individuals with atypical male or female phenotype are to be considered to have a "disorder of sexual development." The goal is to eliminate previous terminology that included the terms intersex, hermaphrodite, or pseudohermaphrodite. However, the teaching of embryology, and particularly teaching about the development of the reproductive system, still has not made the change to the new terminology. If those who teach embryology to health-care professionals remain unaware of the controversies associated with the old terminology and continue to use it, they will perpetuate a nomenclature that can be destructive. Any terminology must be used carefully to avoid dehumanizing the individual to a disease or a medical state. We should be able to state clearly the variations in morphology that exist, attend to the immediate health of the individual, and avoid any attempt to stigmatize gender-atypical individuals.

Fetal Sex Results of Noninvasive Prenatal Testing and Differences With Ultrasonography.

OBJECTIVE: To assess the causes of reported discordance between noninvasive prenatal testing (NIPT) and ultrasound or other clinical information. METHODS: In this retrospective, observational study, all cases in which single-nucleotide polymorphism (SNP)-based NIPT reported normal sex chromosomes and the laboratory was notified by the patient or health care provider of discordance between NIPT and observed or expected fetal sex from clinical information were reviewed. When discordances were unresolved after internal and external laboratory clerical data review or repeat ultrasound imaging, additional clinical records, genetic testing results and pregnancy outcomes were reviewed. RESULTS: Of the 1,301,117 eligible NIPT cases, fetal sex discordances were reported in 91 (0.007%; 1:14,300; 95% CI 1:11,600-1:17,800); partial or complete outcome information was available for 83 of 91 cases. In 30 of 83 (36%) cases, karyotyping was performed, and sufficient clinical information was provided to establish the diagnosis of disorders of sexual development. The disorders of sexual development were classified into three categories: 46,XY disorders of sexual development (n=19), 46,XX disorders of sexual development (n=4), and sex chromosome disorders of sexual development (n=7). In 28 of 83 (34%) cases, the cause of the apparent discrepancy was attributable to human error, predominantly phlebotomy labeling or ultrasound misassignment. In 25 of 83 cases, a diagnosis was not possible; the outcome reported was either abnormal (18/83, 22%) or no abnormalities were reported (7/83, 8%). When normal sex chromosomes were predicted by SNP-based NIPT and clinical information was discordant, disorders of sexual development were common. Internal laboratory clerical data review and re-imaging confirmed the NIPT fetal sex reports in 34% cases, providing reassurance that no further evaluation was necessary. CONCLUSION: Identification of apparent fetal sex discordances with NIPT results, and reporting this suspicion to the laboratory, provides an opportunity for further evaluation to identify the cause of apparent discordances and the involvement of a multi-disciplinary team, as necessary to prepare for postnatal care. We propose a protocol for evaluation of these cases. FUNDING SOURCE: This study was funded by Natera, Inc.

Maternal stress alters plasma testosterone in fetal males.

Titers of testosterone in plasma were determined by radioimmunoassay in male rat fetuses of stressed and control mothers on days 17, 18, 19, 21, and 23 (the day of birth) after conception. In fetuses of stressed mothers, testosterone concentrations were highest on day 17, declined on days 18 and 19, and then remained unchanged. In the control fetuses, testosterone increased from relatively low concentrations on day 17 to the highest amounts on days 18 and 19, and then declined. Thus, the persistence of feminine and impaired masculine sexual behavior in male offspring of stressed mothers could be due to the absence of a surge of circulating testosterone during days 18 and 19 after conception, a period postulated to be critical in the development of the central nervous system in the rat.

Genetically determined sex-reversal in 46,XY humans.

Evidence is presented for the existence of a gene, probably on the X chromosome, which prevents testis differentiation when present in 46,XY human embryos. Affected 46,XY women are not completely normal because of premature ovarian involution, as a result of which they have "streak gonads" similiar to those of 45,X women.

Sex differentiation disorders (SDD) prenatal sonographic diagnosis, genetic and hormonal work-up.

Gender is determined by the genetic, gonadal and hormonal/ phenotypic sex. Genetic sex is determined at conception. The establishment of the gonadal sex (ovary/testis) and the phenotypic sex (external and internal genitalia) is a complicated multistep process which is determined during fetal life mainly during the first trimester. Recently more genes have been found to be involved in this process. Prenatal diagnosis of fetal gender can be made using ultrasound technology, genetic and hormonal examinations. Nowadays using a vaginal and abdominal transducer for US examination recognition of external and internal genitalia of both genders is possible. The determination of gender during fetal life is important not only as a matter of curiosity; in some cases of ambiguity (for example congenital adrenal hyperplasia) prenatal treatment can change the natural history of the disease. Prenatal diagnosis can also subtype the ambiguity, and its severity can be established. In this review we describe our experience in prenatal diagnosis and establishment of the fetal gender, the subtypes of ambiguity and our suggestion for the process of diagnostic work-up.

Revisiting the X:A signal that specifies Caenorhabditis elegans sexual fate.

In Caenorhabditis elegans, sex is determined by the opposing actions of X-signal elements (XSEs) and autosomal signal elements (ASEs), which communicate the ratio of X chromosomes to sets of autosomes (X:A signal). This study delves more deeply into the mechanism by which XSEs transmit X chromosome dose. We determined the relative contributions of individual XSEs to the X:A signal and showed the order of XSE strength to be sex-1 > sex-2 > fox-1 > ceh-39 >/= region 1 XSE. sex-1 exerts a more potent influence on sex determination and dosage compensation than any other XSE by functioning in two separate capacities in the pathway: sex-1 acts upstream as an XSE to repress xol-1 and downstream as an activator of hermaphrodite development and dosage compensation. Furthermore, the process of dosage compensation affects expression of the very XSEs that control it; XSEs become fully dosage compensated once sex is determined. The X:A signal is then equivalent between XO and XX animals, causing sexual differentiation to be controlled by genes downstream of xol-1 in the sex-determination pathway. Prior to the onset of dosage compensation, the difference in XSE expression between XX and XO embryos appears to be greater than twofold, making X chromosome counting a robust process.

Freemartin in the amphibian Pleurodeles waltl: parabiosis between individuals from opposite sex triggers both germ and somatic cells alterations during female gonad development.

Wild type embryos of the newt Pleurodeles waltl were used to realize parabiosis, a useful model to study the effect of endogenous circulating hormones on gonad development. The genotypic sex of each parabiont (ZZ male or ZW female) was determined early from the analysis of the sex chromosome borne marker peptidase-1. In ZZ/ZZ and ZW/ZW associations, gonads develop according to genetic sex. In ZZ/ZW associations, the ZZ gonads differentiate as normal testes while ZW gonads development shows numerous alterations. At the beginning of sex differentiation, these ZW gonads possess a reduced number of germ cells and a reduced expression of steroidogenic factor 1 and P450-aromatase mRNAs when compared to gonads from ZW/ZW associations. During gonad differentiation, conversely to the control situation, these germ cells do not enter meiosis as corroborated by chromatin status and absence of the meiosis entry marker DMC1; the activity of the estradiol-producing enzyme P450-aromatase is as low as in ZZ gonads. At adulthood, no germ cells are observed on histological sections, consistently with the absence of VASA expression. At this stage, the testis-specific marker DMRT1 is expressed only in ZZ gonads, suggesting that the somatic compartment of the ZW gonad is not masculinized. So, when exposed to ZZ hormones, ZW gonads reach the undifferentiated status but the ovary differentiation does not occur. This gonad is inhibited by a process affecting both somatic and germ cells. Additionally, the ZW gonad inhibition does not occur in the case of an exogenous estradiol treatment of larvae.

Sexual differentiation of behavior: the foundation of a developmental model of psychosexuality.

The sexual differentiation of the brain and behavior occurs as the result of prenatal hormonal influences. Knowledge of this area is helpful for the construction of an appropriately modern psychoanalytically informed developmental paradigm of psychosexuality.

Nematode development: evolutionary detours of a pivotal cell.

The anchor cell plays a central role in organizing the reproductive structures of the nematode Caenorhabditis elegans. Recent studies show that significant alterations in the origin, function and fate of this key regulatory cell have occurred during the course of nematode evolution.