WNT7A mutations in patients with Müllerian duct abnormalities.

STUDY OBJECTIVE: WNT7A gene mutations were evaluated as a potential cause for Müllerian duct derivative abnormalities in human females. The WNT gene family encodes glycoproteins that serve as signaling molecules during early development. The WNT7A gene has been previously identified as necessary for normal murine Müllerian duct development. WNT7A mutant mice display several Müllerian duct derivative abnormalities. DESIGN: Molecular genetic analysis of female patients with Müllerian duct derivative abnormalities. SETTING: Medical center-based academic research institution. PARTICIPANTS: 40 women with developmental abnormalities of the uterus and vagina and 12 normal controls. INTERVENTIONS: Polymerase chain reaction DNA amplification from human genomic DNA and denaturing gradient gel electrophoresis analysis of amplified DNA fragments. MAIN OUTCOME MEASURES: Presence or absence of WNT7A gene mutations in analyzed DNA fragments. RESULTS: No mutations were found in the WNT7A gene in any patient or control tested. CONCLUSIONS: WNT7A mutations are an unlikely cause of Müllerian duct derivative abnormalities in humans.

The N314D polymorphism of the GALT gene is not associated with congenital absence of the uterus and vagina.

The aetiology of anomalous embryonic and fetal development of the female reproductive tract, ranging from common uterine abnormalities to the somewhat rare congenital absence of the uterus and vagina (CAUV), is unknown. Some have proposed that abnormal galactose metabolism might cause CAUV. An association between CAUV and the N314D allele of the galactose-1-phosphate uridyl transferase (GALT) gene has been proposed as aetiological. We tested this hypothesis further by performing a case-control molecular study analysing 32 patients with CAUV for the presence of the N314D allele. These patients were compared with 138 normal controls. No association between CAUV and the N314D polymorphism was found (P = 0.32). It is unlikely that either maternal or fetal GALT enzyme activity could affect paramesonephric duct development, because neither galactosaemic subjects nor their children have an increased incidence of uterine anomalies.

Gender bias in the disposition of frozen embryos.

OBJECTIVE: To examine the gender differences found among couples when choosing the disposition of their frozen embryos. DESIGN: Retrospective chart review. SETTING: University affiliated in vitro fertilization (IVF) center. PATIENTS: Couples undergoing their first cycle of assisted reproductive technology (ART). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Choice of disposition of gametes and embryos. RESULT(S): Gender bias is found when couples choose the disposition of their frozen embryos, but not when they choose the disposition of their gametes. CONCLUSION(S): Gender bias was found in couples who made decisions regarding the disposition of their frozen embryos.

Familial gonadotropin-releasing hormone resistance and hypogonadotropic hypogonadism in a family with multiple affected individuals.

OBJECTIVE: To characterize the phenotype of idiopathic hypogonadotropic hypogonadism due to compound heterozygous GnRHR gene mutations (Arg262Gln/Tyr284Cys). DESIGN: Retrospective review. SETTING: Tertiary medical center. PATIENT(S): Family containing four siblings (three female and one male) with complete idiopathic hypogonadotropic hypogonadism. INTERVENTION(S): Baseline and stimulated laboratory studies. One patient received GnRH treatment and one received human menopausal gonadotropins. MAIN OUTCOME MEASURE(S): Clinical phenotype vs. genotype is assessed by endocrine studies, karyotype, pedigree, and review of pathology slides of ovarian neoplasm. RESULT(S): With GnRH stimulation, two patients with idiopathic hypogonadotropic hypogonadism had maximum LH < 10 mIU/mL, and two others had peak LH > 10 mIU/mL. With repeated GnRH stimulation 24 hours later, gonadotropin levels in all patients were increased. Stimulation of thyroid-releasing hormone and tests for insulin-induced hypoglycemia were normal. One affected patient did not ovulate after GnRH treatment, but her sister ovulated with gonadotropin treatment. Another affected sibling had bilateral oophorectomy for seromucinous cystadenomas, and her hypogonadotropic state remained after castration. The man with idiopathic hypogonadotropic hypogonadism and his unaffected brother had a ring chromosome 21. CONCLUSION(S): All patients with complete idiopathic hypogonadotropic hypogonadism had the same GnRHR mutations, but clinical presentations and endocrinologic responses were heterogeneous. Gonadotropin levels remained low in patients with idiopathic hypogonadotropic hypogonadism after castration, and ring chromosome 21 was present, suggesting that sequences from this chromosome could affect the idiopathic hypogonadotropic hypogonadism phenotype.

Role for anti-Müllerian hormone in congenital absence of the uterus and vagina.

Molecular genetic techniques were used to determine if mutations in the genes encoding anti-Müllerian hormone (AMH) (also known as Müllerian inhibiting substance (MIS)) and its receptor (AMHR) are commonly present in patients with congenital absence of the uterus and vagina (CAUV). Twenty-two CAUV patients and 96 control subjects from diverse ethnic groups were studied after obtaining informed consent. Genomic DNA samples prepared from leukocytes were digested separately with several different restriction enzymes, and the resultant fragments were analyzed for restriction fragment melting polymorphisms (RFMPs) by denaturing gradient gel electrophoresis (DGGE). Electrophoretic mobility of DNA fragments which were 200-700 base pairs in length was compared using polyacrylamide gels that included linear gradients of denaturing solvents designed to separate DNA fragments according to sequence-dependent variation in thermal stability. Two RFMPs were found in the AMH gene in both patients and normal control subjects. One RFMP in the AMHR gene was present at low frequencies in both patients and normal control subjects. No RFMPs specific to CAUV patients were found in either gene. Because no mutations or rare DNA sequence polymorphisms were detected in the AMH and the AMHR genes in this group of CAUV patients, it is unlikely that either gene commonly has an etiologic role in CAUV.

Dominant transmission of imperforate hymen.

OBJECTIVE: Imperforate hymen is an uncommon anomaly of the reproductive tract, occurring in approximately 0.1% of newborn females. The familial occurrence of imperforate hymen in a child, her mother, and her mother's monozygotic twin is reported. DESIGN: Case report. SETTING: Academic medical center. PATIENT(S): Three affected family members. MAIN OUTCOME MEASURE(S): Karyotype and pedigree analysis. RESULT(S): The proband, presenting with peritonitis, was evaluated at age 12 for imperforate hymen because this condition was diagnosed in her mother at age 14. At age 14, the mother's monozygotic twin was asymptomatic except for primary amenorrhea and was also demonstrated to have imperforate hymen. No other reproductive system abnormalities were known to be present in the remaining family members. Chromosomal structural analysis confirmed that the mother of the proband had no chromosomal abnormalities. CONCLUSION(S): The occurrence of imperforate hymen in two consecutive generations of a family is consistent with a dominant mode of transmission, either sex-linked or autosomal. Previously reported examples of siblings with imperforate hymen suggested a recessive mode of inheritance. Taken together, these cases suggest that imperforate hymen can be caused by mutations in several genes. This case highlights the importance of evaluating all family members of affected patients. Familial examples of other developmental anomalies of the female reproductive tract also suggest a multifactorial genetic etiology.

Contemporary issues for spontaneous abortion. Does recurrent abortion exist?

Most of the time, spontaneous abortion is a random event and represents the natural selection process. Although a recurrent factor may be present and may cause one or more abortions for a given couple, such instances are rare. Well-substantiated causes include parental chromosomal abnormalities (e.g., translocation), antiphospholipid syndrome, PCOD, and maternal age greater than 40 years. Müllerian duplication defects are most likely a cause of pregnancy loss for some women. A growing body of evidence refutes the role of corpus luteum defect as a common cause of recurrent abortion. Other causes are numerically infrequent in occurrence. It is likely that cigarette smoking and alcohol consumption contribute to pregnancy wastage. Although some therapies for the causes listed herein have been proven effective by randomized controlled trials, most have not. Given the excellent outcome demonstrated for most couples with unexplained recurrent abortion in the absence of treatment, it is difficult to recommend unproven therapies, especially if they are invasive and expensive. Instead of examining the environment in which pregnancy has occurred or been planned, clinicians have simply counted the number of spontaneous abortions among couples in an attempt to determine who should be evaluated. The former approach would seem most appropriate and proactive.

Methylation-dependent melting polymorphisms in genomic fragments of deoxyribonucleic acid.

OBJECTIVES: Denaturing gradient gel electrophoresis can detect single base sequence differences in deoxyribonucleic acid and methylation differences in small cloned fragments of deoxyribonucleic acid. We previously detected cell type-specific melting differences by denaturing gradient gel electrophoresis in paired leukocyte and sperm cell samples of deoxyribonucleic acid. We proposed that these differences were caused by differential methylation and that blotting strategies using denaturing gradient gel electrophoresis might be useful in detecting in vivo variations in methylation patterns. STUDY DESIGN: Genomic deoxyribonucleic acid from leukocytes and sperm cells of 35 male subjects was analyzed by denaturing gradient gel electrophoresis after digestion by 4-bp site enzymes and Msp I and its methylation-sensitive isoschizomer Hpa II. Some fragments were amplified by polymerase chain reaction. RESULTS: Cell type-specific melting polymorphisms were detected in all genes from all subjects. Analysis of Msp I/Hpa II sites demonstrated that differences noted correlated with the methylation state. Cell type-specific differences were absent in fragments amplified by polymerase chain reaction. CONCLUSIONS: The denaturing gradient gel electrophoresis blotting technique is a fast and comprehensive method for comparing in vivo methylation differences.

Administration of progesterone before oocyte retrieval negatively affects the implantation rate.

OBJECTIVE: To compare the efficacy of two clinically accepted methods of progesterone supplementation during IVF. DESIGN: Prospective randomized trial. SETTING: A university-based IVF program. PATIENT(S): Three hundred fourteen stimulated IVF cycles between January 1993 and October 1994. INTERVENTION(S): Patients were assigned to one of two luteal phase progesterone regimens by a random permuted block design. In protocol A, 12.5 mg of IM progesterone was given 12 hours before oocyte retrieval; in protocol B, 25 mg of IM progesterone was given on the day of oocyte retrieval. MAIN OUTCOME MEASURE(S): Clinical pregnancy. RESULT(S): Patient demographic characteristics, including age, diagnosis, number of oocytes retrieved and fertilized, and number of embryos transferred, were not different between the two groups. There was no difference in the rate of cycle cancellation between the groups. One hundred forty ETs were performed in patients assigned to protocol A and 142 in patients assigned to protocol B. The clinical pregnancy rate in group A was 12.9% compared with 24.6% in group B. CONCLUSION(S): The administration of progesterone before oocyte retrieval is associated with a lower pregnancy rate than the administration of progesterone after oocyte retrieval.

Further evidence that the WT1 gene does not have a role in the development of the derivatives of the müllerian duct.

OBJECTIVE: Several lines of evidence suggest that expression of the WT1 transcription factor gene is necessary for normal development of the renal and male reproductive systems. Female patients with severe reproductive tract developmental defects were examined for WT1 gene mutations. STUDY DESIGN: The WT1 gene was analyzed in 25 patients with congenital absence of the uterus and vagina for mutations. Genomic deoxyribonucleic acid prepared from blood leukocytes was subjected to Southern blot analysis and denaturing gradient gel electrophoresis. RESULTS: Common WT1 gene deoxyribonucleic acid sequence polymorphisms were found in both normal control subjects and patients with congenital absence of the uterus and vagina. No deoxyribonucleic sequence differences or mutations likely to cause congenital absence of the uterus and vagina were detected in the patients. CONCLUSIONS: The absence of WT1 gene mutations in patients with congenital absence of the uterus and vagina supports the hypothesis that WT1 expression is required only for later urogenital development, after the mesonephric and paramesonephric ducts have already formed.

Technical and physiological aspects associated with the lower fertilization following intra cytoplasmic sperm injection (ICSI) in human.

The fertilization rates with ICSI range from 30% to 70% and suggest that, despite injecting sperm into mature oocytes, significant fertilization failure still occurs in humans. The objective of this study was to determine technical and physiological factors which may contribute to lower fertilization following ICSI. Eggs that failed to show two pronuclei (PN) 48 hours after ICSI were studied at two different time intervals: at ICSI program inception (group A) and after 8 months (group B). The eggs were analyzed by staining with DNA fluorochromes, Hoescht 33258 and DAPI. The extent of sperm head as well as maternal chromatin decondensation in unfertilized ICSI eggs was determined by high resolution fluorescence microscopy. The average fertilization rate (FR) from all ICSI cycles in these two groups was 45%. The FR in Groups A and B were 35% and 59%, respectively (P < 0.05). In Group A, 65% of the unfertilized eggs were characterized by condensed sperm chromatin with 11% showing partial decondensation. In Group B, only 28% of the unfertilized eggs demonstrated condensed sperm chromatin while 45% were partially decondensed. Sperm chromatin was not detected in 24% of all unfertilized eggs studied. The maternal chromatin remained at metaphase II in 84% of all unfertilized eggs analyzed. These observations suggest that the technical problem of deposition of the sperm inside the egg is not the major cause for failure of fertilization rates in ICSI cycles. The increased percentage of eggs undergoing sperm head decondensation may be related to subtle changes in technique as experience is gained over time. The failure of sperm head decondensation in some of the ICSI eggs may be associated with cytoplasmic immaturity but not nuclear maturity.

Failed fertilization after intracytoplasmic sperm injection: the extent of paternal and maternal chromatin decondensation.

OBJECTIVE: To determine the extent of paternal and maternal chromatin decondensation in unfertilized eggs after intracytoplasmic sperm injection (ICSI). DESIGN: Eggs that failed to show two pronuclei (2-PN) 48 hours after ICSI were studied at two different time intervals: at ICSI program inception (group A) and after 8 months (group B). PATIENT(S): Forty-nine patients undergoing IVF cycles. MAIN OUTCOME MEASURE(S): The unfertilized eggs were studied by chromatin staining. RESULT(S): The average fertilization rate from all ICSI cycles in these two groups was 45%. The fertilization rates in groups A and B were 35% and 59%, respectively. In group A, 65% of the unfertilized eggs were characterized by condensed sperm chromatin with 11% showing partial decondensation. In group B, only 28% of the unfertilized eggs demonstrated condensed sperm chromatin, whereas 45% were partially decondensed. In these two groups, no sperm chromatin was detected in 24% of the unfertilized eggs. The maternal chromatin remained at metaphase II in 84% of all unfertilized eggs analyzed. CONCLUSION(S): These observations suggest that the technical problem of deposition of the sperm inside the egg is not the major cause of failure of fertilization rates in ICSI cycles. Rather, it is likely to be the failure to complete both the maternal and paternal chromatin transitions that occur with normal fertilization.

Human chorionic gonadotrophin-beta gene sequences in women with disorders of HCG production.

Women with recurrent abortion, primary unexplained infertility, and gestational trophoblastic neoplasia (GTN) manifest disordered human chorionic gonadotrophin (HCG) secretion. Mutations in the HCG beta/luteinizing hormone (LH) beta gene complex could cause aberrant HCG production in these disorders. The purpose of this study was to determine whether HCG beta gene deletions occur in women with recurrent abortion or primary unexplained infertility, and whether HCG beta gene duplications are present in women with GTN. DNA was extracted from 10 patients with unexplained recurrent abortion, 10 patients with unexplained primary infertility, 12 patients with GTN, three partners of women with GTN, and 30 controls. Southern blots were constructed and hybridized with DNA probes for HCG beta-5 and the LH beta gene. No gene deletions were identified in patients with recurrent abortion or primary unexplained infertility. Likewise, no gene duplications were identified in women with GTN. A previously described Mbol restriction fragment length polymorphism (RFLP) was identified in both patients and controls. A new Pstl RFLP was also characterized, but was present in patients and controls. Deletion/duplication mutations in the HCG beta/LH beta gene complex do not appear to be common causes of aberrant HCG production in humans with these disorders.

Congenital absence of the uterus and vagina is not commonly transmitted as a dominant genetic trait: outcomes of surrogate pregnancies.

OBJECTIVE: To determine the inheritance pattern of congenital absence of the uterus and vagina in affected women undergoing surrogacy IVF with this disorder. DESIGN: Retrospective study. SETTING: A hospital-based reproductive endocrinology and infertility center. PATIENT(S): Women diagnosed with congenital absence of the uterus and vagina undergoing IVF with subsequent transfer of embryos to a surrogate uterus. INTERVENTION(S): Questionnaires were sent to all infertility treatment centers performing surrogate procedures. MAIN OUTCOME MEASURE(S): Number, gender, and frequency of congenital anomalies in progeny. RESULT(S): Thirty-two of 53 surveyed programs responded (60%). One hundred sixty-two IVF cycles were performed, and 34 liveborn children were delivered (half female). No congenital anomalies were found, except for one male child with a middle ear defect and hearing loss. CONCLUSION(S): These results strongly suggest that congenital absence of the uterus and vagina, if genetically transmitted, is not inherited commonly in a dominant fashion.

Mutation analysis of the gonadotropin-releasing hormone receptor gene in idiopathic hypogonadotropic hypogonadism.

OBJECTIVE: To determine if GnRH receptor mutations occur in patients with idiopathic hypogonadotropic hypogonadism. DESIGN: Patients and controls were studied by molecular genetic analysis. SETTING: A tertiary medical center setting. PATIENT(S): Twenty-four patients with idiopathic hypogonadotropic hypogonadism and 20 controls. INTERVENTION(S): Deoxyribonucleic acid from all individuals was analyzed by Southern blot analysis and denaturing gradient gel electrophoresis. Genomic DNA was digested with restriction enzymes, and Southern blots and denaturing gradient gel blots were constructed. Blots were hybridized with the GnRH receptor complementary DNA probe. The DNA sequencing was performed on samples from two representative patients. MAIN OUTCOME MEASURE(S): Gonadotropin-releasing hormone receptor gene structure was ascertained by comparing fragments from autoradiographs in patients and controls. Individual nucleotides were ascertained from DNA sequencing gels. RESULT(S): No GnRH receptor gene deletions or polymorphisms were identified by Southern blot analysis. New restriction-fragment melting polymorphisms using the enzymes DpnII, RsaI, and HaeIII were identified by denaturing gradient gel blots in patients and controls. CONCLUSION(S): Gonadotropin-releasing hormone receptor gene deletions or rearrangements were not observed in our idiopathic hypogonadotropic hypogonadism patients. Denaturing gradient gel electrophoresis failed to identify single-base differences unique to patients with idiopathic hypogonadotropic hypogonadism, dramatically reducing the likelihood that point mutations of the GnRH receptor gene are present in idiopathic hypogonadotropic hypogonadism.

Ultrasound prediction of follicle volume: is the mean diameter reflective?

OBJECTIVE: To evaluate the relationship between 2 dimensional sonographic measurement of ovarian follicles and their actual volume. DESIGN: Prospective clinical study. SETTING: The in vitro fertilization (IVF) program of a University based, tertiary care hospital. PATIENTS AND INTERVENTIONS: Sonographic categorization by shape, and measurement of 96 individual ovarian follicles immediately prior to aspiration for IVF. Each follicle was aspirated under direct ultrasound guidance and the volume recorded. The 96 follicles were visualized in a total of 14 patients from whom 2 to 27 oocytes were obtained. MAIN OUTCOME MEASURE: Total volume of each follicle. RESULTS: Round and polygonal follicles exhibited a highly significant relationship between sonographically measured mean diameter and total follicle volume. The volume of follicles that were categorized as ellipsoid was not predicted by measurement of the longest diameter, shortest diameter or mean diameter. CONCLUSION: The mean diameter of round and polygonal follicles accurately predicts total follicular volume. However, clinical decisions in ovulation induction should be modified when the follicle shape is predominantly ellipsoid because the traditionally held belief that the sonographic measurement of the follicular diameter correlates with the follicular volume does not apply in those circumstances.

Aberrant estradiol flare despite gonadotropin-releasing hormone-agonist-induced suppression is associated with impaired implantation.

Our results confirm the previous report that rapid suppression by GnRH-a is favorable relative to delayed suppression (1). They further indicate that the pattern of E2 production during GnRH-a-induced ovarian suppression may be predictive of cycle outcome. We suggest that imperfect pituitary suppression of bioactive LH as indicated by an aberrant rise in E2 during GnRH-a down-regulation may compromise oocyte quality and ultimately impair implantation. Further study of follicular phase E2 response to GnRH-a suppression may provide a prognostic marker for implantation.

Gonadotropin-releasing hormone-associated peptide gene sequences in women with hyperprolactinemia.

OBJECTIVE: To determine if mutations in the structural gene for gonadotropin-releasing hormone (GnRH)-associated peptide are present in women with hyperprolactinemia. DESIGN: Patients with hyperprolactinemia and controls were studied retrospectively for GnRH-associated peptide gene mutations. SETTINGS: Patients seen in a clinical setting were studied at a medical school laboratory setting. PATIENTS: Fifteen women with hyperprolactinemia and two fertile controls with normal prolactin levels were studied. INTERVENTIONS: Genomic deoxyribonucleic acid (DNA) was extracted from each patient and subjected to Southern blot analysis and polymerase chain reaction (PCR). For Southern blot analysis, DNA was digested with EcoRI, XbaI, BglII, PstI, and BamHI and hybridized to two DNA probes for GnRH-associated peptide. Exons II to IV, which encode for the structural gene, were amplified by PCR. MAIN OUTCOME MEASURES: Fragment sizes from autoradiographs were compared among patients and controls. Amplified PCR products of exons II to IV of the GnRH-associated peptide were also compared. RESULTS: No large deletions, insertions, or polymorphisms were identified in women with hyperprolactinemia or controls by Southern blotting. Each of the exons was present and of normal size by PCR in the study patients and controls. CONCLUSIONS: No large deletions of the GnRH-associated peptide gene appear to be present in our patients with hyperprolactinemia. Small deletions, insertions, or point mutations are not excluded by this analysis.