Estil Strawn

Promoter Hypermethylation of Progesterone Receptor Isoform B (PR-B) in Endometriosis

The physiological effects of progesterone (P) is mediated by two isoforms of progesterone receptors (PRs): PR-A and PR-B. Progestins have long been used in the treatment of endometriosis but unfortunately the relief of pain is relatively short-term. In addition, about 9% of women with endometriosis simply do not respond to progestin therapy due to reasons unknown. In fact, a general tendency for relative progesterone resistance within eutopic and ectopic endometrium of women with endometriosis and the downregulation of PR-B, but not PR-A, in endometriosis have been noted. Since promoter hypermethylation is well-documented to be associated with transcriptional silencing, we sought to determine the methylation status of the PR-A and PR-B promoter regions in the epithelial component of endometriotic implants using a combination of laser capture microdissection (LCM), methylation specific PCR, and bisulfite sequencing. We found that the promoter region of PR-B, but not PR-A, is hypermethylated in endometriosis as compared with controls. In addition, the PR-B expression was significantly reduced in the ectopic endometrium. Our finding suggests that progesterone resistance in endometriosis in general and the down-regulation of PR-B, but not PR-A, in particular, are a result of promoter hypermethylation of PR-B, but not PR-A. This, in conjunction with our reported aberrant methylation of HOXA10 in the eutopic endometrium of women with endometriosis, strongly suggests that endometriosis is an epigenetic disease. This perspective should potentially open up new avenues for the delineation of pathogenesis of endometriosis, and might also lead to novel ways to treat the disease through reversing aberrant methylation via pharmacological means.

Resolution of clonal origins for endometriotic lesions using laser capture microdissection and the human androgen receptor (HUMARA) assay

OBJECTIVE To determine the clonal origins of endometriotic lesions using laser capture microdissection and PCR-based HUMARA assay. DESIGN Molecular genetic study of human tissue. SETTING Molecular genetics laboratory in an academic setting. PATIENT(S) Twenty patients with endometriosis. Forty specimens of endometriotic lesions from these patients and one specimen of normal endometrium were analyzed. INTERVENTION(S) Laser capture microdissection was used to harvest epithelial cells from single and multifocal endometrial lesions from paraffin-embedded and frozen tissues, and their clonality was determined with the HUMARA assay. MAIN OUTCOME MEASURE(S) Polymerase chain reaction-based HUMARA assay of clonality. RESULT(S) Thirty-eight specimens were polymorphic and thus informative. Most specimens were monoclonal, as determined by the HUMARA assay. In four specimens of multifocal lesions, polyclonality was detected, but upon more refined microdissections and further analyses, we found that each focus was monoclonal individually. CONCLUSION(S) Previously reported polyclonality is very likely to be attributed to the pooling of multifocal lesions or contamination of normal tissues. These results suggest that endometriotic lesions were monoclonal in origin, and in the case of multifocal lesions, each focus originates monoclonally; hence, different foci have independent origins. The monoclonality of endometriotic lesions suggests that they may carry neoplastic potentials, and the apparent independent origins of multifocal lesions suggest that reconstruction of individual lesion histories may help us to understand the initiation and progression of endometriosis.

Genomic alterations in ectopic and eutopic endometria of women with endometriosis.

Background/Aims: Ectopic and eutopic endometria of women with endometriosis have been shown to contain genomic alterations. In this study, we sought to identify genomic alterations in both ectopic and eutopic endometria of 5 women with endometriosis and to examine whether the two tissues share any genomic alterations. We also attempted to classify tissue samples based on the alteration profiles. Methods: Laser capture microdissection was used to harvest epithelial cells. High-resolution comparative genomic hybridization microarrays were used to identify genomic alterations in eutopic and ectopic endometria from 5 women with endometriosis. The results were validated by real-time RT-PCR and loss of heterozygosity analysis. Results: All 5 patients had genomic alterations in their eutopic and ectopic endometria. The ectopic and eutopic endometria shared a sizable portion of genomic alterations. Cluster analysis of the genomic alteration profile correctly and consistently classified tissue samples from the 5 patients into two groups: peritoneal implants and ovarian cysts. Conclusions: The correct classification of tissue samples into two groups suggests that these two subtypes of endometriosis may have distinct genomic alteration profiles and are thus distinct entities, as previously proposed. The shared alterations are likely the ones that harbor genes responsible for an increased propensity of endometrial debris to implant to the ectopic sites and for early events that lead to the establishment of lesions. Alternatively, these shared alterations may harbor genes that are dysregulated in both eutopic and ectopic endometria. The identified genomic alterations should help to zero in genes involved in the pathogenesis of endometriosis in future studies.

Birth of a healthy infant following preimplantation PKHD1 haplotyping for autosomal recessive polycystic kidney disease using multiple displacement amplification.

PurposeTo develop a reliable preimplantation genetic diagnosis protocol for couples who both carry a mutant PKHD1 gene wishing to conceive children unaffected with autosomal recessive polycystic kidney disease (ARPKD).MethodsDevelopment of a unique protocol for preimplantation genetic testing using whole genome amplification of single blastomeres by multiple displacement amplification (MDA), and haplotype analysis with novel short tandem repeat (STR) markers from the PKHD1 gene and flanking sequences, and a case report of successful utilization of the protocol followed by successful IVF resulting in the birth of an infant unaffected with ARPKD.ResultsWe have developed 20 polymorphic STR markers suitable for linkage analysis of ARPKD. These linked STR markers have enabled unambiguous identification of the PKHD1 haplotypes of embryos produced by at-risk couples.ConclusionsWe have developed a reliable protocol for preimplantation genetic diagnosis of ARPKD using single-cell MDA products for PKHD1 haplotyping.

Impact of sperm morphology on the likelihood of pregnancy after intrauterine insemination

OBJECTIVE To determine whether there is a difference in ongoing pregnancy rates (PRs) between patients undergoing IUI with strict sperm morphology ≤4% compared with >4% on initial semen analysis. DESIGN Retrospective chart review with multivariate analysis. SETTING Academic outpatient reproductive center. PATIENT(S) A total of 408 couples with male and/or female factor infertility and known strict sperm morphology (SSM). INTERVENTION(S) A total of 856 IUIs with partners sperm (IUI-P). MAIN OUTCOME MEASURE(S) Ongoing PRs based on ultrasound documentation of intrauterine pregnancy with fetal heart tones. RESULT(S) There is no statistically significant difference in per cycle PRs when comparing patients with a strict sperm morphology of ≤4% versus >4% who undergo IUI-P (17.3% vs. 16.7%; odds ratio 0.954, 95% confidence interval 0.66-1.37). Multiple potential confounding factors were assessed using multivariate analysis. CONCLUSION(S) Strict sperm morphology ≤4% is not associated with lower PRs in couples undergoing IUI-P, and thus should not be the sole reason for advancing to IVF.

What is the optimal follicular size before triggering ovulation in intrauterine insemination cycles with clomiphene citrate or letrozole? An analysis of 988 cycles

OBJECTIVE To determine the optimal size of the leading follicle before human chorionic gonadotropin (hCG) administration in cycles with clomiphene citrate (CC) and letrozole, and to examine any differences in the optimal leading follicle size between cycles with CC and letrozole. DESIGN A retrospective study. SETTING University hospital-based reproductive center. PATIENT(S) 1,075 women undergoing intrauterine insemination cycles with CC or letrozole. INTERVENTION(S) Leading follicle diameters and endometrial thickness were recorded 24 hours before hCG administration, together with other cycles parameters, and were compared between pregnant and nonpregnant patients. MAIN OUTCOME MEASURE(S) Leading follicle diameter and intrauterine insemination outcome. RESULT(S) Eight percent of patients (n = 87) were excluded because their leading follicle was less than 18 mm by days 11 to 13. Pregnancy was recorded as clinical pregnancy with fetal heart activity seen at 6- to 7-week transvaginal ultrasound. For both CC and letrozole, higher pregnancy rates were achieved when the leading follicles were in the 23 to 28 mm range. The optimal size of the leading follicle was not statistically significantly different between cycles using CC or letrozole. However, for each endometrial thickness, the optimal follicular size of the leading follicle was different. Each additional millimeter of endometrial thickness increased the optimal follicular size by 0.5 mm. Thicker endometrial lining led to a higher probability of pregnancy. CONCLUSION(S) The optimal size of the leading follicle in ovulation induction with CC and letrozole is similar for both drugs and is closely related to the endometrial thickness.

Is it the patient or the IVF? Beckwith-Wiedemann syndrome in both spontaneous and assisted reproductive conceptions

OBJECTIVE To describe two children diagnosed with Beckwith-Wiedemann Syndrome (BWS) arising from a spontaneous conception and an assisted reproductive technology (ART) cycle from one patient with a long-standing history of subfertility. DESIGN Case report. SETTING Academic medical center. PATIENT(S) Two children with the morphologic features of BWS as a result of a spontaneous conception and an ART cycle from the same patient. INTERVENTION(S) Assisted reproductive technology. MAIN OUTCOME MEASURE(S) Neonatal and pediatric morphologic evaluation by geneticists. RESULT(S) Two children with the morphologic features consistent with the criteria for the diagnosis of BWS. CONCLUSION(S) Patients with subfertility may be carriers for genetic disorders that can be passed to a child with or without the use of assisted reproductive technologies (ART). The use of ART may bypass natural selection mechanisms.

Preimplantation HLA haplotyping using tri-, tetra-, and pentanucleotide short tandem repeats for HLA matching

PurposeTo aid couples wishing to conceive children who are HLA matched to a sibling in need of a hematopoietic progenitor cell transplant, we developed a preimplantation HLA haplotype analysis of embryos that utilizes tri-, tetra-, and pentanucleotide STR markers.MethodsFor preimplantation HLA genotyping, we use polymorphic STR markers located across the HLA and flanking regions, selecting exclusively tri-, tetra-, and pentanucleotide repeats. These markers can be resolved using either capillary electrophoresis (CE) or polyacrylamide gels.ResultsWe have developed 43 reliable STR markers for preimplantation HLA matching. Selected STR markers enabled unambiguous identification of embryos whose HLA haplotypes were matched with the affected patient using polyacrylamide gel or capillary electrophoresis.ConclusionsThe use of tri-, tetra-, and pentanucleotide repeat markers and polyacrylamide gels for STR genotyping in HLA matching is a simple and cost effective approach to clinical testing.

Isolated abnormal strict morphology is not a contraindication for intrauterine insemination.

This study sought to investigate whether isolated abnormal strict morphology (<5% normal forms) and very low strict morphology (0–1% normal forms) affects pregnancy rates in intrauterine insemination (IUI). This was a retrospective study performed at an Academic Medical Center/Reproductive Medicine Center. Four hundred and eight couples were included for 856 IUI cycles. 70 IUI cycles were performed in couples with abnormal strict morphology and otherwise normal semen parameters. Outcomes were measured as clinical pregnancy rate per IUI cycle as documented by fetal heart activity on maternal ultrasound. Clinical pregnancy rate did not significantly differ between the group with abnormal strict morphology [11/70 (15.7%)] and the normal morphology group [39/281 (13.9%)]. Additionally, there was no significant difference between the pregnancy rate in the abnormal morphology group compared to that of our overall institutional IUI pregnancy rate [145/856 (16.9%)]. Furthermore, there was no significant difference between pregnancy rate in the very low morphology group [3/14 (21.4%)] compared to those with normal morphology or the overall IUI pregnancy rate. Patients with isolated abnormal strict morphology have clinical pregnancy rates similar to those with normal morphology for IUI. Even in those with very low normal forms, consideration of IUI for assisted reproduction should not be excluded.

Outcomes After Testicular Aspiration and Testicular Tissue Cryopreservation for Obstructive Azoospermia and Ejaculatory Dysfunction

PURPOSE We examined the rate of successful sperm retrieval, fertilization, pregnancy and live births in patients with obstructive azoospermia and ejaculatory dysfunction who underwent testicular sperm aspiration in conjunction with in vitro fertilization/intracytoplasmic sperm injection using cryopreserved testicular tissue. MATERIALS AND METHODS We retrospectively reviewed the records of 40 couples who underwent testicular sperm aspiration from August 2003 to November 2007. All procedures were performed before intracytoplasmic sperm injection with the goal of tissue cryopreservation. RESULTS Mature sperm was successfully retrieved and cryopreserved in 39 of 40 patients (97%) with an average of 5 vials cryopreserved per couple. Of the women 34 underwent in vitro fertilization/intracytoplasmic sperm injection and in 5 treatment was planned. The fertilization rate was 58% with a pregnancy rate of 61.4% (27 of 44 cycles). There were 17 live births, 5 ongoing pregnancies and 5 miscarriages. No complications were noted and all patients returned to full activity within 2 to 3 days. CONCLUSIONS Testicular sperm aspiration with cryopreservation is a safe and effective sperm retrieval method in patients with obstructive azoospermia and ejaculatory dysfunction when in vitro fertilization/intracytoplasmic sperm injection will be performed.