A mathematical model was developed to calculate the implantation probability for individual embryos based on the pregnancy outcome of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cases with multiple embryos transferred. This model was used to calculate implantation probabilities of embryos of 31 morphological types using the outcome of 1,200 IVF/ICSI cases. The algorithm was validated by comparing the calculated pregnancy probability and multiple pregnancy probability with the actual outcome of 281 separate IVF/ICSI cases. Finally, an estimation of embryo synergy was calculated.

The growth differentiating factor-9 (GDF-9) gene is expressed specifically in oocytes and plays an important role in folliculogenesis. GDF-9 knock-out female mice develop ovarian failure. Homozygous mutations in the sheep GDF9 gene are also associated with a disruption of normal follicle development and infertility. Our objective was to determine if GDF-9 mutations were present in our patients with premature ovarian failure (POF). Mutation screening in prospectively collected samples of women with POF. Sixty-two women with idiopathic POF ascertained from different geographical regions in the United States, have been recruited since 2001 for the Baylor College of Medicine POF study. Idiopathic POF was defined as two serum follicle stimulating hormone concentrations greater than 20 IU/ml before age 40 in the absence of known chromosomal aneuploidy. Sixty women without a history of POF comprised our control group. Genomic DNA was extracted from leukocytes and both exons of GDF-9 amplified separately using polymerase chain reaction (PCR) with specific primers for each exon. PCR products for each exon were denatured and reannealed to form potential heteroduplexes (wild-type strand paired with mutant strand). Homozygous samples formed heteroduplexes when they were mixed with wild-type DNA. Heteroduplexes were detected using denaturing high-performance liquid chromatography (DHPLC) on the WAVE System 3500 (Transgenomic Ltd, Omaha, NE). Samples which demonstrated heteroduplex formation on DHPLC were then sequenced directly after PCR amplification on an automated sequencer, ABI PRISM 310 (Applied Biosystems, Foster City, CA). Exon 1 of GDF-9 was abnormal in one woman who developed POF at age 22; her medical history was negative for autoimmune disorders and there was no family history of POF. Her karyotype was 46,XX. DNA sequencing showed heterozygous substitution which resulted in an altered amino acid from proline (P) to serine (S) at residue 103 (P103S). This amino acid change occurs in a highly conserved proprotein region in several species (Table 1) and plausibly should affect the cleavage of proprotein to mature protein. Although studies of other family members are obviously necessary, no heteroduplexes were found in the other 61 cases or in the 60 control women. Analysis of exon 2 demonstrated heteroduplexes in approximately 70% of POF cases; however, three known single nucleotide polymorphisms (SNP) accounted for these cases. Tabled 1 GDF-9 plays an important role in follicle development. Surprisingly, perturbations are uncommon in women with POF as a P103S mutation was observed in only one out of 62 cases.

Objective: To investigate quantitative aberrations involving p53 copy numbers in eutopic endometrial and endometriotic tissue from two populations.Design: Comparative analysis of normal and diseased tissue.Setting: Tissue specimens collected in Iceland and USA.Patient(s): Subjects with moderate/severe endometriosis (Iceland, n = 26; USA, n = 45). Paraffin-embedded tissue from 19 matched Icelandic cases and seven unaffected controls. American cases were fresh surgical tissue from 17 matched cases and 28 unaffected controls. DNA isolation and real-time polymerase chain reaction (PCR) with TaqMan assay were performed.main Outcome Measure(s): The frequency of p53 loss and/or gain based on quantitative differences for copy numbers of p53 located on chromosome (17p) and GAPDH on a control locus (chromosome 12p).Result(s): Among American cases, significant p53 gain (n = 13) or loss (n = 4) was observed in 17 of 21 cases. In Icelandic cases this was not seen to the same degree. Mean normalized p53 values were 3.46 and 1.16 copies per reaction, respectively. Significant differences were observed between normalized p53 in the control blood and affected tissue for the American and Icelandic cases compared to standard GAPDH control but not in normal Icelandic and American endometrium.Conclusion(s): The results continue to support a role for nonrandom somatic p53 locus alterations in the pathogenesis of late or sever-stage endometriosis. Differences between Icelandic and American subjects have implications for generalization of genome-wide approaches.

ObjectiveWomen undergoing hysterectomy with oophorectomy report significant decrease in sexual desire compared to women who undergo hysterectomy alone. This difference has been attributed to decreased androgen concentrations resulting from ovarian removal. We therefore designed this prospective controlled trial to compare androgen concentrations in oophorectomized women compared to those not undergoing oophorectomy at hysterectomy. In addition, the study was conducted over a two-year period of time to determine if the differences were sustained or even more profound over time in oophorectomized women.DesignProspective case control studyMaterials and methodsFourteen women who were scheduled to undergo hysterectomy with or without oophorectomy were recruited for the study. Nine women had ovarian conservation and 5 women had oophorectomy. Women who had oophorectomy received estrogen 0.05 mg transdermally. Hormone analysis using a validated RIA, with preassay chromatography was performed preoperatively, 1, 3, 6, 12, 18 and 24 months after surgery. Analyses were performed using mixed-model ANOVA, with group, oophorectomy versus not, as the between-subject factor, time as the within subject factor and outcome as percentage of baseline value at each time point.ResultsThere was an immediate and statistically significant decline in total testosterone, free testosterone, and androstenedione in the oophorectomized group compared to the ovarian conservation group. The difference between groups varied from 20-50%. There was no statistically significant difference in total testosterone, free testosterone and androstenedione over time for subjects in both groups. There was no significant difference in SHBG and DHEAS between the groups.ConclusionsIn these late premenopausal women, we have demonstrated that oophorectomy results in an immediate decline in testosterone and multiple other circulating androgens. This decline is sustained but does not become more profound over 2 years. We conclude that diminished sexual desire reported in oophorectomized women compared to ovarian intact women may be the result of losses in circulating ovarian androgens. ObjectiveWomen undergoing hysterectomy with oophorectomy report significant decrease in sexual desire compared to women who undergo hysterectomy alone. This difference has been attributed to decreased androgen concentrations resulting from ovarian removal. We therefore designed this prospective controlled trial to compare androgen concentrations in oophorectomized women compared to those not undergoing oophorectomy at hysterectomy. In addition, the study was conducted over a two-year period of time to determine if the differences were sustained or even more profound over time in oophorectomized women. Women undergoing hysterectomy with oophorectomy report significant decrease in sexual desire compared to women who undergo hysterectomy alone. This difference has been attributed to decreased androgen concentrations resulting from ovarian removal. We therefore designed this prospective controlled trial to compare androgen concentrations in oophorectomized women compared to those not undergoing oophorectomy at hysterectomy. In addition, the study was conducted over a two-year period of time to determine if the differences were sustained or even more profound over time in oophorectomized women. DesignProspective case control study Prospective case control study Materials and methodsFourteen women who were scheduled to undergo hysterectomy with or without oophorectomy were recruited for the study. Nine women had ovarian conservation and 5 women had oophorectomy. Women who had oophorectomy received estrogen 0.05 mg transdermally. Hormone analysis using a validated RIA, with preassay chromatography was performed preoperatively, 1, 3, 6, 12, 18 and 24 months after surgery. Analyses were performed using mixed-model ANOVA, with group, oophorectomy versus not, as the between-subject factor, time as the within subject factor and outcome as percentage of baseline value at each time point. Fourteen women who were scheduled to undergo hysterectomy with or without oophorectomy were recruited for the study. Nine women had ovarian conservation and 5 women had oophorectomy. Women who had oophorectomy received estrogen 0.05 mg transdermally. Hormone analysis using a validated RIA, with preassay chromatography was performed preoperatively, 1, 3, 6, 12, 18 and 24 months after surgery. Analyses were performed using mixed-model ANOVA, with group, oophorectomy versus not, as the between-subject factor, time as the within subject factor and outcome as percentage of baseline value at each time point. ResultsThere was an immediate and statistically significant decline in total testosterone, free testosterone, and androstenedione in the oophorectomized group compared to the ovarian conservation group. The difference between groups varied from 20-50%. There was no statistically significant difference in total testosterone, free testosterone and androstenedione over time for subjects in both groups. There was no significant difference in SHBG and DHEAS between the groups. There was an immediate and statistically significant decline in total testosterone, free testosterone, and androstenedione in the oophorectomized group compared to the ovarian conservation group. The difference between groups varied from 20-50%. There was no statistically significant difference in total testosterone, free testosterone and androstenedione over time for subjects in both groups. There was no significant difference in SHBG and DHEAS between the groups. ConclusionsIn these late premenopausal women, we have demonstrated that oophorectomy results in an immediate decline in testosterone and multiple other circulating androgens. This decline is sustained but does not become more profound over 2 years. We conclude that diminished sexual desire reported in oophorectomized women compared to ovarian intact women may be the result of losses in circulating ovarian androgens. In these late premenopausal women, we have demonstrated that oophorectomy results in an immediate decline in testosterone and multiple other circulating androgens. This decline is sustained but does not become more profound over 2 years. We conclude that diminished sexual desire reported in oophorectomized women compared to ovarian intact women may be the result of losses in circulating ovarian androgens.

Many couples are now seeking preimplantation genetic diagnosis (PGD) and fluorescence in-situ hybridization (FISH) as an alternative approach to avoid spontaneous abortion by ensuring transfer of presumed chromosomally normal embryos. This case report describes unexpected findings in a couple having three spontaneous abortions and two failed IVF cycles. In two IVF PGD cycles, four of 13 (30.8%) embryos (blastomeres) demonstrated duplication involving the Down syndrome critical region, detectable by a locus specific chromosome 21 probe. The same duplication was subsequently detected by FISH in 66 of 1002 (6.6%) sperm nuclei, demonstrating paternal gonadal mosaicism. Cytogenetic studies of peripheral blood revealed normal karyotypes in both the male and female partners. This identification of paternal germ cell or gonadal mosaicism suggests that analysis of sperm nuclei prior to undergoing IVF with PGD may be of value in patients with recurrent spontaneous abortions or multiple failed IVF.

BACKGROUND:Spatial organization of chromosomes is hypothesized to reflect transcriptional activity and regulatory protein function. Preimplantation genetic diagnosis allows assessment of the spatial relationship of chromosomes in human blastomeres. We thus examined the localization of chromosomes 13, 16, 18, 21, 22, X and Y in blastomeres from 6-8-cell stage embryos, correlating localization to aneuploidy and embryo morphology.METHODS:Following fluorescence in situ hybridization to enumerate chromosomes 13, 16, 18, 21, 22, X and Y, signal positions were localized within one of four concentric shells. Statistical analysis compared chromosome localization between euploid and aneuploid blastomeres as well as morphologically normal and abnormal embryos.RESULTS:Of 98 embryos, 109 blastomeres were evaluated. Within chromosomally normal blastomeres, no difference in the location of all seven chromosomes (P</=0.10) was observed. However, a significant difference was observed between the organization of chromosomes in euploid versus aneuploid blastomeres (P</=0.001). Localization of chromosomes 13, 18, 21 and 22 was significantly different when an abnormality involving that chromosome existed (P</=0.001, P</=0.01, P</=0.025 and P</=0.01 respectively).CONCLUSIONS:We report for the first time that localization of chromosomes is altered in chromosomally aneuploid but not in chromosomally normal nor morphologically abnormal euploid blastomeres.

To determine the chromosomal complement of ectopic pregnancies that fail methotrexate therapy by comparative genomic hybridization Examination of archived ectopic pregnancy tissue by comparative genomic hybridization (CGH). Seventeen patients were identified that demonstrated surgically confirmed tubal rupture after MTX therapy. Pathology tissue blocks were obtained on 17 identified patients. Gestational age, means of conception (spontaneous versus assisted reproductive technology), ultrasound findings, history of ectopic pregnancy, history of sexually transmitted disease, age, gravidity, parity, time from first MTX to rupture, and surgical implantation site were recorded from patients' charts. Human chorionic gonadotropin (hCG) levels and serial trends were recorded in detail. Genomic anlysis was performed by comparative genomic hybridization and restricted to the chorionic villi using coning cyclindars. Of the 17 tissue blocks available, 3 blocks did not contain villi, and were therefore not amenable for study. In fifteen cases, DNA was successfully extracted from the microdissected tissues and was of sufficient quantity and quality for evaluation by CGH after degenerate oligonucleotide-primed PCR. Eleven of fifteen were euploid. Four of fifteen ectopic gestations were aneuploid. Three of the four aneuploid gestations were mosaic, with normal 46,XX cell lines present. The four aneuploid gestations did not differ clinically from the euploid gestations in terms of patient age, gravidity, history of ectopic pregnancy or pelvic adhesive disease, gestational age at diagnosis, hCG at diagnosis, peak hCG, days to operative management, or peak hCG change (Pyr>0.05 for all parameters). In this sample, the majority of ectopic pregnancies that fail methotrexate are euploid. Our results also confirm the feasibility of CGH for archived tissues.

BACKGROUND A biochemical marker for embryo development would increase the chance of a successful pregnancy with IVF by optimizing oocyte and embryo selection, and allow fewer embryos to be transferred. In this study, we correlated cumulus granulosa cell gene expression of hyaluronic acid synthase 2 (HAS2), cyclooxygenase 2 (COX2; PTGS2) and gremlin (GREM1) with subsequent embryo development in search of a parameter for embryo selection. METHODS Cumulus cell gene expression was determined prospectively on eight consecutive patients undergoing IVF with ICSI. Immediately following oocyte retrieval, the cumulus was stripped from the oocyte, and cumulus gene expression for PTGS2, HAS2 and GREM1 was assessed using a one-step real-time quantitative RT-PCR assay. Oocyte quality, fertilization and embryo morphology were correlated to relative gene expression. RESULTS Gene expression data were available on cumulus cells from 108 oocytes that developed into 70 embryos (64.8% fertilization rate). Cumulus PTGS2, HAS2 and GREM1 expression was higher from oocytes that developed into higher quality embryos (grades 3, 4 and 5) compared with lower quality embryos (grades 1 and 2) (P<0.05, P<0.001 and P<0.001, respectively). HAS2 and GREM1 expression was also higher from the cumulus surrounding oocytes that gave rise to higher grade embryos (P<0.001). The expression of PTGS2 and HAS2 was 6-fold higher, and that of GREM1 was 15-fold higher in cumulus yielding higher grade embryos versus lower grade embryos. CONCLUSION PTGS2, HAS2 and GREM1 gene expression correlates to morphological and physiological characteristics and provides a novel approach to predict human embryo development. Ultimately, with better predictors of follicular and embryonic health, higher quality embryos can be selected and transferred, reducing higher order pregnancy rates.

Objective: To assess the pregnancy outcome of IVF with intracytoplasmic sperm injection (ICSI) in couples with profound teratospermia (Kruger's strict criteria of zero).Design: Retrospective analysis of 545 consecutive cycles of IVF/ICSI performed between January 2000 and January 2003.Setting: Tertiary care center.Patient(s): Of 545 IVF/ICSI cycles, 45 patients were identified with a semen strict morphology of 0 using Kruger's strict criteria.Intervention(s): Ovarian down-regulation (Lupton) was followed by controlled ovarian stimulation exclusively with hMG. Embryo transfer was performed 2 days after transvaginal aspiration/ICSI.Main Outcomes Measure(s): Pregnancy outcomes and newbom/infant status.Result(s): Of 45 patients undergoing 54 treatment cycles, 21 patients were positive for pregnancy (38.9% pregnancy/cycle). No birth defects were noted at time of delivery and all infants had obtained appropriate developmental milestones at I year of age.Conclusion(s): Men with profound teratospermia (Kruger's strict criteria of zero) may achieve acceptable pregnancy rates after IVF/ICSI thereby alleviating the use of donor sperm in this group. Furthermore, no increased risk of birth defects is apparent in this small series. (C) 2004 by American Society for Reproductive Medicine.

DNA methylation at promoter regions usually results in transcriptional repression of the downstream gene. An age-dependent increase in DNA methylation at the promoter region of the estrogen receptor alpha gene (ERα) has been demonstrated in several human tissues such as colon, atrium and fibroblasts. Our objective was to determine whether an age-dependent increase in DNA methylation of the ERα also occurs in human granulosa cells. Prospective pilot study on 9 patients undergoing assisted reproductive technology at Baylor College of Medicine. Patient age, number of oocytes retrieved and estradiol level at the time of human chorionic gonadotropin trigger were recorded. All patients underwent a standard ovarian stimulation protocol with gonadotropin releasing hormone agonist (Lupron®) downregulation followed by controlled ovarian hyperstimulation using human menopausal gonadotropins. Granulosa cells were isolated and frozen at -80oC immediately following oocyte retrieval. DNA was extracted using a commercial DNA extraction kit. All DNA samples were treated with sodium bisulfite. The cytosine-phosphoguanine (CpG) island containing region of the ERα promoter was amplified by polymerase chain reaction (PCR) with primers specific to bisulfite modified DNA. During bisulfite modification and PCR amplification, unmethylated cytosines are first converted to uracil then to thymine. PCR products were purified, isolated and cloned into a plasmid vector using commercially available kits. Sequencing was performed on an automated sequencer. The unmodified CpG to overall CpG dinucleotide ratio (methylation ratio) was calculated. Correlations between patient age, number of oocytes, estradiol level and methylation ratio were determined using Spearman's correlation coefficient. The median age was 35 (range 22–41). The median methylation ratio was 1.8% (range 0.45–2.5%). There was no significant correlation between patient age and methylation ratio (P>0.05). Neither estradiol levels nor number of oocytes retrieved correlated with methylation ratio (P>0.05). •There was no age-dependent increase in the DNA methylation of the ERα promoter region. • This region was minimally methylated in human granulosa cells from patients who underwent controlled ovarian hyperstimulation. • Our study demonstrates the feasibility of this technique to examine the methylation status of candidate genes in human granulosa cells.

The American Society for Reproductive Medicine (ASRM) membership survey, although subject to response bias, provided clear opinions on controversial issues. A past president and the current president of ASRM comment on the value of the input to the ASRM board. (C) 2004 by American Society for Reproductive Medicine.

Objective: To evaluate the effect of physiologic oral DHEA (dehydroepiandrosterone) administration on exercise tolerance in a group of postmenopausal women with low endogenous DHEAS levels. Design: Randomized, double-blinded, parallel, placebo-controlled trial with a dose titration protocol. The trial lasted one year. Materials and Methods: Nineteen postmenopausal women were randomized to DHEA treatment (n=9) and placebo (n=10), given at 8: 00 am. The treatment group was given a 40 mg. daily dose of oral micronized DHEA (courtesy Belmar pharmacy, Lakewood CO) for one year, with monthly dose titration by an unblinded investigator to maintain levels of DHEA-S between 300 and 450 microgram/dL. The subjects were non smokers who were not on HRT. Among other parameters, standard aerobic exercise tolerance testing was performed at baseline and 1 year, measuring peak heart rate (PHR) and peak volume O2 consumption (VO2 peak). At the time of exercise testing, resting and peak cardiac outputs (QT-R and QT-P) were measured by a rebreathing technique. Statistical assessment was by ANOVA. Results: Post treatment VO2 peak, a measure of maximum aerobic capacity, was increased in the DHEA treatment group as compared to the placebo group (24.49 ± 1.24 ml/kg/min vs.18.35 ± 0.79; means ± SEM, P=0.019). Further, the change in VO2 peak increased in the DHEA group, compared to a decrease in the placebo group (2.27 0.94 ml/kg/min vs.-1.15 ± 0.64; means ± SEM, P=0.01). However, no significant differences were noted between the groups in cardiac output (at rest or during exercise) or peak heart rate. Conclusion: Physiologic dose-titrated replacement of DHEA in postmenopausal women with low endogenous DHEAS levels appears to improve the maximum volume of oxygen consumed by the body each minute during exercise, one measure of capacity for aerobic work. This change is seen without other changes in exercise parameters that would indicate a central cardiac inotropic effect, although numbers are small. This preliminary data would support a peripheral effect of DHEA or its metabolites on improving exercise performance, perhaps at the level of muscle. We have previously demonstrated that DHEA supplementation may improve insulin sensitivity, and these findings may be a partial reflection of that change. In all, DHEA supplementation in postmenopausal women may possibly partially counteract the natural decline in aerobic capacity that occurs with aging.

Unexplained infertility is estimated to occur in 10%–15% of couples seeking fertility treatment (1Crosignani P.G. Collins J. Cooke I.D. Diczfalusy E. Rubin B. Recommendations of the ESHRE workshop on "unexplained infertility." Anacapri, August 28–9, 1992.Hum Reprod. 1993; 8: 977-980PubMed Google Scholar) and is a diagnosis by exclusion. We propose that a genetic mutation in the progesterone receptor (PROGINS), long known to be essential for reproduction, may have an etiologic role in some couples with heretofore-unexplained infertility. This mutation results in an amino acid substitution in the hinge region of the progesterone receptor and alters the function of the receptor (2Kieback D.G. Tong X.W. Wiegel N.L. Agoulnick I.U. A genetic mutation in the progesterone receptor (PROGINS) leads to an increased risk of nonfamilial breast and ovarian cancer causing inadequate control of estrogen receptor driven proliferation [Abstract no. 6].Gynecologic Investigation. 1998; : 40aGoogle Scholar). This functional alteration and the significance of the progesterone receptor in reproduction led us to evaluate this mutated allele in a female population with unexplained infertility. Twenty-six Caucasian women with the diagnosis of unexplained infertility were included in this study. This study was approved by the Institutional Review Board. Criteria for the diagnosis of unexplained infertility and inclusion consisted of the following: [1] infertility for >1 year duration; [2] females 23 to 40 years of age; [3] 25- to 34-day ovulatory cycles confirmed by urinary ovulation testing; [4] normal luteal phase endometrial biopsy; [5] normal day 3 FSH; [6] normal serum TSH; [7] normal serum prolactin; [8] normal intrauterine cavity evaluated by either hysterosalpingogram or hysteroscopy; and [9] normal pelvic peritoneum and fallopian tubes evaluated by laparoscopy. Eleven subjects with minimal or mild endometriosis were included. Male partners had [1] normal semen analysis (3World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. 2–19. Cambridge University Press: New York, 1992.Google Scholar) and [2] absence of anti-sperm antibodies detected by immunobead testing in the seminal plasma. The 28 Caucasian controls consisted of fertile women with at least one full-term birth and no history of infertility. Subjects underwent up to four cycles of controlled ovarian hyperstimulation and intrauterine insemination. Patients received either FSH (Metrodin [Serono Laboratories, Norwell, MA] or Fertinex [urofollitropin, Serono]) or hMG (Pergonal [Serono] or Humagon [Organon, West Orange, NJ]). In addition to controlled ovarian hyperstimulation and insemination, four women underwent one cycle of IVF. Genomic DNA was isolated from the buffy coat as described elsewhere (4McKenna N.J. Kieback D.G. Carney D.N. Fanning M. McLinden J. Headon D.R. A germline TaqI restriction fragment length polymorphism in the progesterone receptor gene in ovarian carcinoma.Br J Cancer. 1995; 71: 451-455Crossref PubMed Scopus (75) Google Scholar). A 10-μL polymerase chain reaction (PCR) mixture was prepared containing 4 μM of each intron G primer, sense: 5′-TGAAGTTGTTTGACAAGCTGTTGG-3′ and antisense: 5′-GCCTCTAAAATGAAAGGCAGAAAGC-3′; 250 μM of each dNTP; 100 ng of genomic DNA; 0.2 units of Taq polymerase; 5 mM KCl; 1 mM Tris-HCl (pH 8.3); and 0.15 mM of MgCl2. The cycling profile consisted of denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute, with an initial denaturation step at 94°C for 3 minutes. This was run for a total of 34 cycles. After PCR, 10 μL of amplified product was subjected to electrophoresis on a 1.5% agarose gel stained with ethidium bromide. The 320–base pair (bp) Alu insertion in Intron G produces a 494-bp fragment compared with a 174-bp fragment present in wild types (Fig. 1A). Because all three mutations occur simultaneously (2Kieback D.G. Tong X.W. Wiegel N.L. Agoulnick I.U. A genetic mutation in the progesterone receptor (PROGINS) leads to an increased risk of nonfamilial breast and ovarian cancer causing inadequate control of estrogen receptor driven proliferation [Abstract no. 6].Gynecologic Investigation. 1998; : 40aGoogle Scholar), restriction fragment length polymorphism analysis was carried out on exon 5 for confirmation. A 10-μL PCR mixture was prepared in the same fashion as for intron G, except primers for exon 5 were used, sense: 5′-GTTTTCGAAACTTACATATTG-3′ and antisense: 5′-TCATTTAGTATTAGATCAGGT-3′, and the annealing temperature was decreased to 54°C. The PCR product was subject to digestion with the restriction enzyme NlaIII (New England Biolabs, Beverly, MA) according to the manufacturers' recommendation. Ten microliters of the digested product was subjected to electrophoresis on a 1.5% agarose gel stained with ethidium bromide. The C to T mutation creates an additional enzyme recognition site for NlaIII (CATG). Compared with wild type with a single 159-bp fragment, homozygous mutants contain 2 fragments: 106 and 53 bp. Subjects heterozygous for the allele contain all three fragments: 159, 106, and 53 bp (Fig. 1B). Fisher's exact test was used to compare the prevalence of the PROGINS allele between the infertile group and controls. Student's t test was used to compare age of conception and weight at first pregnancy. Fisher's exact test was used to compare family history of ovarian or breast cancer and pregnancy rates. The Kruskal-Wallis test was used to compare the subjects' ages, cycle 1 peak estradiol levels, cycle 1 estradiol per follicle, number of mature follicles, number of ampules of medication used, and day of hCG administration. Eleven of 26 (42%) women with unexplained infertility were heterozygous for PROGINS. This was significantly increased over controls, among whom only 4 of 28 (14%) women were heterozygous for PROGINS. There were no homozygous mutants in either group. Infertility patients were older than controls (31.8 ± 5.62 years of age vs. 24.2 ± 5.62 years of age). Although there was a statistically significant difference in the age of our cases vs. that of the controls, the overall ages were young when considering age as a factor in infertility. There was no statistically significant difference in weight at conception, family history of ovarian cancer, or family history of breast cancer. In the group of women with unexplained infertility, clinical parameters of the stimulation cycle and outcomes were compared in women who carry PROGINS and in those women with unexplained infertility who do not carry the allele. There was no statistically significant difference in the subjects' ages, cycle 1 peak estradiol levels, cycle 1 estradiol per follicle, number of mature follicles, number of ampules of medication used, and day of hCG administration. There was also no statistically significant difference in pregnancy rates. A progesterone receptor mutation, PROGINS, is significantly more prevalent in women with unexplained infertility than in fertile controls. In this study, 42% of women with unexplained infertility carried the mutant allele compared with 14% of fertile controls, a statistically significant increase. To our knowledge, this is the first report describing a human progesterone receptor mutation in association with infertility. This mutated allele consists of an Alu insertion at intron G, a G to T point mutation in the first nucleotide of codon 660 within exon 4 resulting in a valine to leucine substitution, and a C to T point mutation in the third nucleotide of codon 770 within exon 5 resulting in a silent base pair change (2Kieback D.G. Tong X.W. Wiegel N.L. Agoulnick I.U. A genetic mutation in the progesterone receptor (PROGINS) leads to an increased risk of nonfamilial breast and ovarian cancer causing inadequate control of estrogen receptor driven proliferation [Abstract no. 6].Gynecologic Investigation. 1998; : 40aGoogle Scholar). This amino acid substitution in exon 4, a region encoding the hinge region, is an area involved in receptor dimerization (5Tetel M.J. Jung S. Carbajo P. Ladtkow T. Skafar D.F. Edwards D.P. Hinge and amino terminal sequences contribute to solution dimerization of human progesterone receptor.Mol Endocrinol. 1997; 11: 1114-1128Crossref PubMed Scopus (62) Google Scholar), transcriptional activation (6Tsai M. O'Malley B.W. Molecular mechanisms of action of steroid/thyroid receptor superfamily members.Annu Rev Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2666) Google Scholar), nuclear localization (7Guiochon-Mantel A. Loosfelt H. Lescop P. Sar S. Atger M. et al.Mechanisim of nuclear localization of the progesterone receptor evidence for interaction between monomers.Cell. 1989; 57: 1147-1154Abstract Full Text PDF PubMed Scopus (240) Google Scholar), ligand binding (5Tetel M.J. Jung S. Carbajo P. Ladtkow T. Skafar D.F. Edwards D.P. Hinge and amino terminal sequences contribute to solution dimerization of human progesterone receptor.Mol Endocrinol. 1997; 11: 1114-1128Crossref PubMed Scopus (62) Google Scholar), and interactions with co-repressors (8Jackson T.A. Richer J.K. Bain D.L. Takimoto G.S. Tung L. Horwitz K.B. The partial agonist activity of antagonist-occupied steroid receptors is controlled by a novel hinge domain-binding coactivator L7/SPA and the corepressors N-CoR or SMRT.Mol Endocrinol. 1997; 11: 693-705Crossref PubMed Scopus (383) Google Scholar). Thus, a mutation in this region might alter overall function. Preliminary data reveal that the PROGINS allele produces a progesterone receptor that has increased transcriptional activity and stability (2Kieback D.G. Tong X.W. Wiegel N.L. Agoulnick I.U. A genetic mutation in the progesterone receptor (PROGINS) leads to an increased risk of nonfamilial breast and ovarian cancer causing inadequate control of estrogen receptor driven proliferation [Abstract no. 6].Gynecologic Investigation. 1998; : 40aGoogle Scholar). Increased receptor stability and transcriptional activity may account for the patients' infertility. Normally, the progesterone receptor is down-regulated in endometrial epithelium during the luteal phase, at a time when embryo implantation takes place (9Lessey B.A. Killiam A.P. Metzger D.A. Haney A.F. Greene G.L. McCartry Jr, K.S. Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle.J Clin Endocrinol Metab. 1988; 67: 334-340Crossref PubMed Scopus (587) Google Scholar). With increased stability and transcriptional activity of the PROGINS receptor, the normal down-regulation of the progesterone receptor at a critical time during implantation may be delayed. Lessey et al. (10Lessey B.A. Yeh I. Castelbaum A.J. Fritz M.A. Ilesanmi A.O. Korzeniowski P. et al.Endometrial progesterone receptors and markers of uterine receptivity in the window of implantation.Fertil Steril. 1996; 65: 477-483PubMed Google Scholar) demonstrated that the elevation of epithelial progesterone receptor is associated with absent expression of the integrin αυβ3 vitronectin receptor, a marker of endometrial receptivity. This can occur despite a histologically normal endometrium, referred to as a type II defect, seen in 39% of women with unexplained infertility and an in-phase endometrium (11Lessey B.A. Castelbaum A.J. Sawin S.W. Sun J. Integrins as markers of uterine receptivity in women with primary unexplained infertility.Fertil Steril. 1995; 63: 535-542Abstract Full Text PDF PubMed Google Scholar), statistics similar to the prevalence of the mutated allele in our infertile patient population. Another marker of implantation, pinopode formation, is also dependent on the downregulation of the progesterone receptor (12Stavreus-Evers A. Nikas G. Sahlin L. Eriksson H. Landgren B. Formation of pinopodes in human endometrium is associated with the concentrations of progesterone and progesterone receptors.Fertil Steril. 2001; 76: 782-791Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Thus, the increased stability of the mutated receptor may lead to altered expression of the integrin αυβ3 vitronectin receptor and pinopode formation despite a normal luteal phase biopsy. Hence, an associated implantation defect in these women with the PROGINS allele may exist. Further investigation of the endometrium of these women is warranted.

Endometriosis long has been recognized as showing heritable tendencies, with recurrence risks of 5% to 7% for first-degree relatives. The risk indicates that polygenic and multifactorial etiology is far more likely to be the cause than mendelian inheritance. The current task is to determine the number and location of genes responsible for endometriosis. Molecular advances of the past decade make identification and elucidation of these genes a reality. The authors review the basis for concluding that endometriosis is a genetic disorder of polygenic/multifactorial inheritance. Genome-wide strategies for identifying causative genes are considered and available data on association or linkage to putative candidate genes systematically reviewed.

This report describes preimplantation genetic diagnosis (PGD) of a couple with a known paternally‐derived balanced cryptic translocation 46,XY.ish t(2q;17q)(210E14−,B37c1+;B37c1−,210E14+) in embryos from a couple who previously had a child with severe mental retardation and was previously described in this journal [Bacino et al., 2000 ]. This child inherited the unbalanced product of translocation from her father: 46,XX.ish der(2)t(2q;17q)pat(210E14−,B37c1+). The couple desired a normal offspring and sought PGD to avoid clinical pregnancy termination. They were treated three times with in vitro fertilization followed by PGD. Two sequential FISH hybridizations were performed. In the first hybridization, telomeric probes to 2q and 17q and a chromosome 17 centromere probe were employed. The second hybridization screened for maternal age‐related aneuploidy (X,Y,13,18,21). Of the 18 informative embryos, only 4 (22%) were normal. The remaining 12 (67%) were abnormal; most with unbalanced products (10/12) from the paternally‐derived rearrangement. The most frequent mode of segregation observed for this cryptic translocation was adjacent‐1 (7/18, 39%). This suggests cryptic translocations are amenable to PGD and, as are traditional translocations, demonstrate higher frequencies of unbalanced segregants than the empiric risk of 10–15% observed at amniocentesis or chorionic villus sampling. Thus, cryptic translocations presumably behave like overt translocations, in that PGD must be performed on a relatively large number of embryos to assure even 2–3 transferable embryos. © 2003 Wiley‐Liss, Inc.

Objective: Whole chromosomes occupy discrete territories within the nucleus for which distinct nuclear addresses have previously been described in cultured lymphocytes and fibroblasts. The spatial organization of chromosomes is hypothesized to reflect transcriptional activity and function of regulatory proteins. Previous studies have shown that chromosomes high in gene density are preferentially localized within the nuclear interior, in contrast to gene-poor chromosomes which localize to the exterior. Thus, altered nuclear arrangement of chromosomes may correspond with abnormal growth and/or cellular division. Preimplantation genetic diagnosis (PGD) has been employed to identify abnormal embryos based on the number of chromosomes or copies of a specific gene. The spatial relationship of chromosomes in human blastomeres has not been previously evaluated. We sought to examine the localization of chromosomes 13, 18, 16, 21, 22, X and Y in blastomeres from 6–8 cell stage embryos identified as either chromosomally normal or abnormal. Design: Retrospective analysis of chromosome localization in human preimplantation embryos Methods and Materials: PGD was performed on 17 couples undergoing in vitro fertilization (IVF) for the following indications: multiple IVF failure (n=7), known structural chromosomal defects (n=5), recurrent pregnancy loss (n=2), X-linked disease (n=1), known mosaism (n=1), and prior child with trisomy 13 (n=1). Embryos were biopsied at the 6–8 cell stage (day 3) and fluorescent in situ hybridization (FISH) was performed. Blastomere nuclei were analyzed using two sequential hybridizations to enumerate chromosomes 13, 18, 21, X, Y (first hybridization) and then 16 and 22 (second hybridization). FISH images were captured and analyzed using imaging software (Applied Imaging(r)). Chromosome positioning based on FISH signals was designated to one of 4 concentric shells (1–4) of equal area from the center of the nucleus to the periphery. Statistical analysis was performed to compare the localization of each chromosome within normal and abnormal blastomeres. Results: Overall, 1010 FISH signals were evaluated from 98 biopsied embryos. Within the normal blastomeres, no difference in the localization of chromosomes 13, 16, 18, 21, 22, X and Y (chi-square=26.07; p≤ 0.10; df=18) was observed. However, a statistically significant difference between the organization of chromosomes in normal versus abnormal blastomeres (chi-square=36.81; p≤ 0.001; df=3) was detected. Among the abnormal blastomeres, the nuclear position of chromosomes was frequently confined to the peripheral region of the nucleus. In comparison to normal blastomeres, the localization of chromosomes 13, 18, 21, and 22 was significantly different when an abnormality involving that chromosome was observed [chi-square= 18.13, p≤.001, df= 3; 12.61, p≤ 0.01, df=3; 9.46, p≤0.25, df=3; 13.08, p≤0.01, df=3; respectively] but not for 16 [chi-square= .85, p≤1.0, df=3]. Conclusions: For the first time, we have shown that localization of chromosomes is altered in normal versus abnormal blastomeres. Evidence of abnormal chromosomal organization implies that the mechanism regulating this phenomenon may be altered in infertile couples. Further investigation is warranted to elucidate the underlying mechanism associated with altered localization of chromosomes in preimplantation embryos.