Molecular analysis of implantation.

The great frontier in reproductive medicine is implantation biology. The scientific understanding of normal implantation lags behind what is known in areas such as fertilization. For obvious reasons this process is difficult to analyze. Recent investigations utilizing molecular biology approaches have scratched the surface of the interaction between the developing trophoblast and the receptive endometrium. This article describes these approaches with techniques such as serial analysis of gene expression (SAGE) and complementary DNA array hybridization, which will soon reveal the key mediators of normal implantation and result in an understanding of failed implantation.

Steroid 5alpha-reductase 2 gene melting polymorphisms in male subjects with azoospermia or oligospermia.

OBJECTIVE: This study was designed to test the hypothesis that mutations in the gene for type 2 steroid 5alpha-reductase (SRD5A2) may be the cause of a phenotype characterized primarily by oligospermia or azoospermia. STUDY DESIGN: Deoxyribonucleic acid from control subjects and subjects with oligospermia (n = 12) and azoospermia (n = 6) were evaluated for mutations in SRD5A2. Methods used for mutation analysis included polymerase chain reaction, Southern blotting, and denaturing gradient gel electrophoresis. RESULTS: Denaturing gradient gel electrophoresis of all 5 amplified exons resulted in similar migration patterns in samples from both control and study subjects. Genomic deoxyribonucleic acid subjected to denaturing gradient gel electrophoresis after restriction digest revealed melting polymorphisms. Direct sequencing of the gene in a single patient with a unique melting polymorphism yielded a normal sequence. CONCLUSIONS: Melting polymorphisms for SRD5A2 were detected in a group of patients with oligospermia or azoospermia. Sequence analysis did not demonstrate functional mutations in the coding sequence of this gene.

Temporal and cell-specific gene expression by human endometrium after coculture with trophoblast.

OBJECTIVE: Our purpose was to identify temporal and stage-specific expression of endometrial genes during coculture with trophoblast cells. STUDY DESIGN: Endometrial stromal cells were cultured to confluence in the presence of estradiol and progesterone. During these culture conditions the gene expression of 1 tissue specimen that secreted abundant prolactin (415 ng/mL culture medium at 21 days) was compared with a second specimen that did not. These 2 tissues were coincubated with trophoblast tissue in a specialized coculture flask. After 4 and 24 hours of culture messenger ribonucleic acid was extracted and reverse transcribed, and the complementary deoxyribonucleic acid products were amplified by polymerase chain reactions. The reverse transcriptase-polymerase chain reaction products were separated by electrophoresis, and potentially important complementary deoxyribonucleic acid fragments were reamplified, inserted into a plasmid vector, and sequenced after recovery. Sequences were submitted for Basic Local Alignment Search Tool searches of GenBank. RESULTS: We observed up-regulation of 6 gene fragments in decidualized endometrium after 4 hours of coculture with choriocarcinoma-derived trophoblast BeWo cells, but only 1 gene fragment was up-regulated after 24 hours of exposure. Conversely, 2 fragments were down-regulated in decidualized stroma that was exposed to BeWo for 4 hours and 2 fragments were underexpressed after the 24-hour exposure. In the parallel experiment stromal cells that failed to secrete prolactin did not elicit the same regulation of expression. The nondecidualized endometrium overexpressed 1 gene fragment after 4 hours of BeWo exposure and overexpressed 4 gene fragments after exposure to BeWo for 24 hours. Underexpression of gene products also occurred with the nondecidualized endometrium, and we observed 2 fragments and 1 fragment to be underexpressed after 4 and 24 hours of BeWo exposure, respectively. To date, 3 of the candidate differential display fragments from these experiments have been cloned and sequenced. An up-regulated fragment (C6225J4EB-1) was 99% identical (167/168 sequences) to a reported nonredundant expressed sequence tags isolated from muscle, brain, ovary, testis, liver, and pregnant uterus tissues. A second up-regulated fragment (C4375J4EB-1) matched 100% identity (117/117) with a reported gene fragment in the expressed sequence tags database of GenBank that was derived from fetal heart and pregnant uterus. Additional characterization of these expressed sequence tags has not been reported. The third up-regulated fragment (C4250J24EB-2) was 100% identical (265/265) to human reduced nicotinamide adenine dinucleotide dehydrogenase III in the nonredundant gene database of GenBank. CONCLUSION: This report demonstrates the potential usefulness that endometrial-trophoblast coculture and differential display can offer for the molecular analysis of implantation phenomena. We have recognized both overexpression and underexpression of interesting gene fragments during the early phases of endometrial responses to paracrine regulators derived from BeWo trophoblast cells. These responses appear to be specific to the degree of endometrial transformation (decidualization) before challenge by the trophoblast and to the duration of the BeWo exposure. Sequence data identified 1 gene with an unidentified function, another gene with a known function, and a fragment not previously recognized. We submit that our model of endometrial-trophoblast coculture offers a novel tool to test cellular responses during implantation, and differential display represents a sensitive technique that can identify many of the important elements of genomic signaling during nidation.

Paternal somatic and germ-line mosaicism for a sex-determining region on Y (SRY) missense mutation leading to recurrent 46,XY sex reversal.

OBJECTIVE: To determine the etiology for recurrent 46,XY sex reversal in a family with two Swyer siblings. DESIGN: Deoxyribonucleic acid (DNA) from peripheral lymphocytes and sperm were analyzed for duplication of the dosage sensitive sex locus (DSS) and for mutations in sex-determining region on Y (SRY). SETTING: An academic teaching hospital. PATIENTS: A family consisting of mother, father, and five phenotypic daughters, of which two were 46,XY sex-reversed females. INTERVENTION: Deoxyribonucleic acid (DNA) extraction, polymerase chain reaction (PCR), Southern blotting, dosage densitometry, single-strand conformation polymorphism (SSCP), and sequencing. MAIN OUTCOME MEASURE: Comparison of control and subject DNA. RESULTS: Deoxyribonucleic acid (DNA) analysis of SRY in genomic DNA from the 46,XY sex-reversed siblings revealed identical missense mutations (T-->G) in both sisters. Analysis of the SRY gene in paternal lymphocyte and sperm DNA revealed mosaicism for wild and mutant (T-->G) SRY sequences. SRY analysis of sperm DNA also demonstrated the same mosaicism for the T-->G missense mutation. CONCLUSION: A postembryonic SRY mutation gave rise to paternal mosaicism for two distinct cell populations (SRY+/SRY-). The presence of a wild type SRY in the somatic cell line may account for a normal pattern of male sexual differentiation, whereas the presence of a mutated SRY in the germ line resulted in two 46,XY sex-reversed offspring. These results confirm a proposed mechanism for the condition of recurrent 46,XY sex-reversed females.

Ovarian endometriomas do not adversely affect pregnancy success following treatment with in vitro fertilization.

PURPOSE: Ovarian endometriomas have an uncertain impact on outcome following in vitro fertilization (IVF). Some authors describe a poor response to ovulation induction, and others observe decreased pregnancy success rates. Conversely, IVF outcomes similar to those of patients undergoing IVF for tubal-factor infertility have also been reported. To determine the impact of ovarian endometriomas on pregnancy success in our IVF program, we identified patients with endometriosis and compared outcomes that were stratified by the presence or absence of an endometrioma at the time of follicular aspiration. METHODS: One hundred eight patients with a diagnosis of endometriosis treated with IVF were identified, retrospectively. In this group, 24 patients completed 29 cycles in which an ovarian endometrioma was aspirated at the time of oocyte retrieval, and 84 patients without endometriomas completed 147 cycles. The cycles from these two groups were compared for differences in peak estradiol, number of mature follicles, number of oocytes, number of embryos transferred, and clinical pregnancies. RESULTS: There were no significant differences between the two groups with respect to peak estradiol, mature follicles, number of oocytes, number of embryos transferred, or clinical pregnancies. CONCLUSIONS: From this retrospective observational analysis it appears that aspiration of an endometrioma at the time of oocyte retrieval has no adverse effect on outcome. This information may prove helpful when faced with the decision to cancel an IVF treatment cycle in patients with this uncommon complication.

Ovarian cholelithiasis after laparoscopic cholecystectomy associated with chronic pelvic pain.

OBJECTIVE: To report a case of chronic pelvic pain associated with ovarian cholelithiasis and discuss prevention and management of this condition. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 39-year-old woman who presented with right lower abdominal pain after a laparoscopic cholecystectomy. INTERVENTION(S): Diagnostic laparoscopy followed by laparotomy with lysis of adhesions and removal of three to four dozen gallstones. MAIN OUTCOME MEASURE(S): Patient's subjective report of pain. RESULT(S): Resolution of patient's pain. CONCLUSION(S): Gallstones spilled into the peritoneal cavity may migrate and adhere to the dependent portions of the pelvis, potentially resulting in pelvic pain or infertility. This suggests the importance of removing inadvertently spilled gallstones at the time of surgery or using nonsurgical methods of gallstone management in reproductive-aged females.

Nerve root compression by herniated intradiscal gas. Case report.

Intervertebral disc degeneration of any etiology may be associated with the formation of spaces or clefts within the disc. Gas collects within these spaces and can be seen roentgenographically. A case is presented in which intradiscal gas herniated into a connective tissue capsule, displacing the left S-1 nerve root and producing symptoms and signs identical to those of a herniated nucleus pulposus. The pathophysiology of gas within a disc space and the possibility that it may herniate much like the nucleus pulposus is discussed.