Association of the eukaryotic vaginal virome with prophylactic antibiotic exposure and reproductive outcomes in a subfertile population undergoing in vitro fertilisation: a prospective exploratory study.

OBJECTIVE: The objective of this study was to use high-throughput sequencing to describe the vaginal eukaryotic DNA virome in patients undergoing in vitro fertilisation (IVF) to examine associations between the vaginal virome, antibiotic exposure and IVF outcomes. DESIGN: Prospective exploratory study. SETTING: Single academic fertility centre. POPULATION: Subfertile women age 18-43 years undergoing their first IVF cycle with a fresh embryo transfer. METHODS: The primary exposure was prophylactic azithromycin or no azithromycin before IVF. A mid-vaginal swab was obtained at the time of embryo transfer for virome analysis. MAIN OUTCOME MEASURES: The primary outcomes compared between exposure groups were characteristics of vaginal virome and clinical pregnancy rates. Secondary outcomes were virome associations with number of oocytes retrieved, number of blastocysts and implantation rate. RESULTS: Twenty-six women contributed a vaginal swab before embryo transfer. There were no significant differences in IVF outcomes between azithromycin groups. There was no association between viral diversity and clinical pregnancy overall. A higher diversity of herpesviruses and α-papillomaviruses was observed in samples from the azithromycin-treated group compared with the no azithromycin group (P = 0.04). In women that received azithromycin, viral diversity was higher in the group that did not achieve clinical pregnancy compared with those who did (P = 0.06). CONCLUSIONS: We demonstrate that the vaginal eukaryotic virome in women undergoing IVF is associated with antibiotic exposure. Additionally, we demonstrate an inverse trend between viral diversity and pregnancy, with a higher number of viruses detected associated with failure to achieve clinical pregnancy in the azithromycin group. TWEETABLE ABSTRACT: Higher viral diversity is associated with prophylactic antibiotic exposure in subfertile women undergoing IVF.

HLA-G genotypes and pregnancy outcome in couples with unexplained recurrent miscarriage.

HLA-G is a non-classical human leukocyte antigen expressed primarily in fetal tissues at the maternal-fetal interface. This expression pattern is unique among HLA genes and suggests that HLA-G may be involved in interactions that are critical in establishing and/or maintaining pregnancy. To evaluate the role of polymorphisms at this locus in maternal-fetal interactions, 113 couples with unexplained recurrent miscarriage were genotyped for seven polymorphisms that define 12 HLA-G alleles. Logistic regression analysis was used to assess whether HLA-G genotypes were associated with an increased risk for a subsequent miscarriage. The presence of an HLA-G*0104 or HLA-G*0105N allele in either partner was significantly associated with an increased risk for miscarriage, after adjustment for maternal age, number of previous miscarriages, history of a previous liveborn, and treatment with paternal mononuclear cells. The *0104 and *0105N alleles are defined by polymorphisms in the alpha-2 domain and encode protein variants that are present only in the full-length HLA-G1 protein. The significant genotype-specific risk in this population suggests that allelic variation in the alpha-2 domain of the HLA-G1 isoforms contributes to recurrent miscarriage.

Reduction of ovarian torsion 1 week after embryo transfer in a patient with bilateral hyperstimulated ovaries.

OBJECTIVE: To report a rare case of unilateral ovarian torsion 1 week after embryo transfer in a patient with bilateral hyperstimulated ovaries. DESIGN: Case report and literature review. SETTING: Reproductive Endocrine division in a university teaching hospital. PATIENT(S): Infertility patients undergoing IVF-ET. INTERVENTION(S): Laparoscopic reduction of adnexa 1 week after ET. MAIN OUTCOME MEASURE(S): Successful preservation of the affected adnexa. RESULT(S): Delivery of 3.324 kg male infant with preservation of the affected ovary. CONCLUSION(S): Untwisting of the affected ovary at laparoscopy without aspiration reduction of cystic masses is appropriate. The outcome of the pregnancy (even very early) in patients with torsion of the adnexa may be favorable after a laparoscopic unwinding of the affected adnexa.

Fertility treatment in the forty and older woman.

PURPOSE: To determine the outcomes and logical progression of fertility treatment in women forty years and older using their own oocytes. METHODS: This was a retrospective study in which 401 completed treatment cycles in 152 women aged forty and older were reviewed. RESULTS: Assisted reproductive technology (ART) cycles (n = 58) were reviewed, comprising both in vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT). Intrauterine insemination (IUI) cycles (n = 343) were reviewed, consisting of 38 unstimulated natural cycle-IUI (NC-IUI), 194 clomiphene citrate-IUI (CC-IUI), and 111 injectable gonadotropins-IUI (INJ-IUI) cycles. The live birth rate of 15.5% for ART cycles was significantly higher than the live birth rate of 3.2% seen for all IUI cycles (p = 0.0007). There were no differences among treatment groups in spontaneous abortion, preterm delivery, or ectopic pregnancy rates. CONCLUSIONS: For women > or = 40 years of age who wish to use their own eggs, ART offers the best chances for conception and delivery.

Mononuclear-cell immunisation in prevention of recurrent miscarriages: a randomised trial.

BACKGROUND: Couples with unexplained recurrent miscarriage may have an alloimmune abnormality that prevents the mother from developing immune responses essential for the survival of the genetically foreign conceptus. Immunisation with paternal mononuclear cells is used as a treatment for such alloimmune-mediated pregnancy losses. However, the published results on this treatment are conflicting. In this study (the Recurrent Miscarriage [REMIS] Study), we investigated whether paternal mononuclear cell immunisation improves the rate of successful pregnancies. METHODS: Women who had had three or more spontaneous abortions of unknown cause were enrolled in a double-blind, multicentre, randomised clinical trial. 91 were assigned immunisation with paternal mononuclear cells (treatment) and 92 immunisation with sterile saline (control). The primary outcomes were the inability to achieve pregnancy within 12 months of randomisation, or a pregnancy which terminated before 28 weeks of gestation (failure); and pregnancy of 28 or more weeks of gestation (success). Two analyses were done: one included all women (intention to treat), and the other included only those who became pregnant. FINDINGS: Two women in each group received no treatment, and eight (three treatment, five control) were censored after an interim analysis. In the analysis of all randomised women who completed the trial, the success rate was 31/86 (36%) in the treatment group and 41/85 (48%) in the control group (odds ratio 0.60 [95% CI 0.33-1.12], p=0.108). In the analysis of pregnant women only, the corresponding success rates were 31/68 (46%) and 41/63 (65%; odds ratio 0.45 [0.22-0.91], p=0.026). The results were unchanged after adjustment for maternal age, number of previous miscarriages, and whether or not the couple had had a previous viable pregnancy. Similar results were obtained in a subgroup analysis of 133 couples with no previous livebirth. INTERPRETATION: Immunisation with paternal mononuclear cells does not improve pregnancy outcome in women with unexplained recurrent miscarriage. This therapy should not be offered as a treatment for pregnancy loss.

Transmission disequilibrium of maternally-inherited CTLA-4 microsatellite alleles in idiopathic recurrent miscarriage.

To elucidate the mechanisms that facilitate tolerance at the maternal-fetal interface, we are investigating the role of genes that are involved in peripheral self-tolerance in couples with idiopathic recurrent miscarriage. CTLA-4 is a negative regulator of T-cell proliferation and has been associated with human autoimmune disease. An AT(n) polymorphism in the 3'-untranslated region (UTR) of the human gene results in AT stretches that vary in length from 16 to 46 bp. We hypothesized that long stretches of AT repeats would result in mRNA instability, and reduced fetal survival in humans. We examined the transmission of AT(n) alleles in 60 couples with a history of > or = 3 unexplained spontaneous abortions to their 51liveborn children and 10 abortuses. The shorter allele was transmitted from heterozygous mothers to 26 of 35 liveborn children (chi2 = 8.3, P = 0.0040) and to three of nine aborted fetuses (chi2 = 1.0, P = 0.317). The shorter allele was transmitted from heterozygous fathers to 15 of 32 liveborn children (chi2 =0.12, P=0.726) and to five of eight aborted fetuses (chi2 = 0.5, P = 0.480). Furthermore, liveborn fetuses who inherited smaller alleles were more likely to represent the first successful pregnancy than liveborn fetuses who inherited larger maternal alleles (Pexact = 0.044) and fetuses of first pregnancies that inherited the smaller allele were significantly more likely to survive to term (Pexact = 0.0086). The preferential transmission of maternally-inherited shorter alleles to liveborn children, but random transmission of paternally-inherited alleles, suggests that CTLA-4 may be imprinted in humans and that this gene may play a role in inducing or maintaining tolerance at the maternal-fetal interface.

Ovulation induction and ovarian malignancy.

Many couples faced with infertility are treated with ovulation inducing medicines. Recently, concerns have been raised about the possible risk of ovarian malignancy after such ovarian stimulation. Theories of pathogenesis for ovarian cancer include incessant ovulation, elevated pituitary gonadotropins, genetic predisposition, and chemical carcinogens. Protective factors include suppression of ovulation, pregnancy, and castration. Low numbers of exposed women who develop ovarian cancer make this a difficult research subject. Research to date demonstrates conflicting results, with some investigators reporting an increased risk of ovarian cancer with fertility drugs, whereas others do not. The likely magnitude of risk, if one believes a risk exists, may be two to three times that of the general population which is at most 4-5% in a woman's lifetime. The present uncertainty makes it challenging to apply this to today's practice of medicine. With continued efforts worldwide, we hope an understanding of this will be forthcoming.

Endometrial thickness is a valid monitoring parameter in cycles of ovulation induction with menotropins alone.

OBJECTIVE: To evaluate the ability of an ultrasound (US)-measured periovulatory endometrial thickness to predict conception in hMG-stimulated cycles. DESIGN: Retrospective. SETTING: A university-based tertiary practice. PATIENTS: One hundred twelve patients undergoing 292 cycles of ovulation induction with hMG alone. MAIN OUTCOME MEASURES: A periovulatory transvaginal US measurement of endometrial thickness was obtained during cycles of ovulation induction with hMG alone. Clinical pregnancy was defined by fetal cardiac activity. Sensitivity and false-positive rates for multiple discriminatory values of endometrial thickness were calculated and a relative operating characteristic (ROC) curve was constructed to evaluate the performance of this test as a predictor of pregnancy. RESULTS: Thirty-eight of 292 cycles resulted in pregnancy. Conception and nonconception cycles showed similar demographics, diagnoses, peak E2, maximum number of follicles, midluteal P, and mean endometrial thickness. Ovulatory dysfunction was a more frequent diagnosis in the conception group. Relative operating characteristic analysis for endometrial thickness as a predictor of pregnancy yielded an area under the curve of 0.623 +/- 0.049 (mean +/- SD). CONCLUSION: Endometrial thickness is a valid screening test for conception outcome in cycles stimulated with hMG. A periovulatory endometrial thickness > or = 10 mm defined 91% of conception cycles. No pregnancy occurred when the endometrium measured < 7 mm.

Does intrauterine insemination offer an advantage to cervical cap insemination in a donor insemination program?

OBJECTIVE: To compare pregnancy outcome after IUI versus cervical cap insemination in a donor insemination program. DESIGN: A randomized prospective clinical trial in which patients were alternately inseminated with cryopreserved human semen using either IUI or cervical cap insemination methods. SETTING: The donor insemination program at Washington University School of Medicine. PATIENTS: Forty-two women with either isolated male factor or male factor plus corrected ovulatory dysfunction using clomiphene citrate underwent 141 cycles of donor insemination. MAIN OUTCOME MEASURES: Clinical pregnancy rates (PRs) defined as a viable intrauterine gestation > 12 weeks or delivered were compared between groups using the chi 2 test. RESULTS: Clinical PRs were significantly higher in the IUI group (16.4%) compared with the cervical cap insemination group (5.9%). The spontaneous abortion rates were similar between the IUI (1.4%) and cervical cap insemination groups (4.4%). CONCLUSIONS: These findings suggest an advantage to IUI over cervical cap insemination in a donor insemination program.

Long-term follow-up of couples after hamster egg penetration testing.

OBJECTIVE: To determine the clinical usefulness of the zona-free hamster egg penetration test as a long-term prognostic indicator for future pregnancy. SETTING: Division of Reproductive Endocrinology and Infertility at the Washington University Medical Center. PARTICIPANTS: All couples (n = 148) who had a hamster egg penetration assay performed between March 1, 1985 and December 31, 1986 were identified and followed with direct or telephone contact up to 68 months after the initial assay. MAIN OUTCOME MEASURE: The monthly fecundity rates using life table analysis and the 5-year incidence of pregnancy were categorized by the percentage of hamster eggs penetrated and by history of previous urologic surgery. RESULTS: There were no significant differences in the rate nor incidence of pregnancy in couples with hamster egg penetration scores of 0%, > 0% and < or = 10%, > 10% and < or = 20%, or > 20%. Although men with previous urologic surgery tended to have lower scores, there was no significant difference in the 5-year incidence of pregnancy. CONCLUSION: The hamster egg penetration score is not predictive of incidence of pregnancy nor time to conception.

Demonstration of specific binding of cocaine to human spermatozoa.

Exposure of males to cocaine has been linked to abnormal development of their offspring. To investigate the possible role of sperm, this study examined the interaction of cocaine with human spermatozoa. Washed sperm were incubated with tritiated cocaine (6.7 nmol/L) with or without unlabeled cocaine (670 mumol/L), and the samples were filtered and the remaining radioactivity quantitated. The specific binding was optimal at 20 minutes and 23 degrees C. Competition studies with tritiated cocaine (3.4 to 66.6 nmol/L) indicated the presence of approximately 3.6 x 10(3) binding sites per cell, with a high affinity receptor dissociation constant (Kd = 12.6 nmol/L). Cocaine concentrations as high as 670 mumol/L had no detectable effect on either the motility or viability of the cells. These results support the hypothesis that the sperm may act as a vector to transport cocaine into an ovum. This novel mechanism could be involved in the abnormal development of offspring of cocaine-exposed males.

Therapeutic donor insemination: a prospective randomized study of scheduling methods.

OBJECTIVE: To compare basal body temperature (BBT) graphs and urinary luteinizing hormone (LH) monitoring in scheduling therapeutic donor insemination. DESIGN: Participants were prospectively randomized to the BBT or LH groups. SETTING: Participants were private patients of the Reproductive Endocrine Division at Washington University School of Medicine. PATIENTS: Inclusion criteria were designed to assure an isolated male factor. Seventy-four of 113 patients completed the study; 18 had ongoing treatment at the end of the study. INTERVENTIONS: Basal body temperature graphs were physician interpreted and appointments prospectively chosen. Luteinizing hormone patients monitored daily urine samples and scheduled an appointment the day after the detected surge. MAIN OUTCOME MEASURES: Fecundity rates, cumulative pregnancy rates, and cost per pregnancy were all prospectively evaluated. RESULTS: Life table analysis yielded a 6-month cumulative probability of pregnancy of 36.3% in the LH group and 65.1% in the BBT group (P less than 0.025). The total cost per pregnancy was lower in the BBT group (+6,212 versus +3,997; P less than 0.001). CONCLUSIONS: This randomized prospective study demonstrates significant therapeutic and economic advantages when therapeutic donor insemination is prospectively scheduled by BBT graphs.

Diisopropyl fluorophosphate labeling of sperm-associated proteinases.

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.

Tumor necrosis factor in peritoneal fluid of women undergoing laparoscopic surgery.

The level of tumor necrosis factor (TNF) in peritoneal fluid (PF-TNF) of 74 women undergoing laparoscopy was determined. The difference between the mean concentration of PF-TNF of women with normal pelvic anatomy and women with moderate or severe endometriosis was significant (P less than 0.01). The proportion of PF-TNF-positive women with PID and those with moderate or severe endometriosis was also significantly higher when compared to women with normal pelvic anatomy (P less than 0.05; P less than 0.02). The proportion of PF-TNF positive women among nulligravid and nulliparous women was significantly higher than that of women with two or more pregnancies (P less than 0.01) and two or more deliveries (P less than 0.005). These results indicate that the presence of PF-TNF is associated with primary infertility and endometriosis.

Pulsatile oxytocin for induction of labor: a randomized prospective controlled study.

In a prospective randomized study, 20 patients with term pregnancies underwent induction of labor with either continuous or pulsed (every 8 minutes) intravenous oxytocin infusion. There were no significant differences with respect to induction-labor interval, induction-delivery interval, cesarean section rates, need for pain relief and Apgar scores. Sixty percent of patients receiving continuous oxytocin infusion developed uterine hyperstimulation but only 10% receiving pulsed oxytocin did so. However, the difference was not significant. The mean +/- SEM total amount of oxytocin given by continuous infusion was 4237 +/- 1066 mU which was 70% more than by pulsatile infusion (2454 +/- 808 mU). The highest rate of oxytocin infused was significantly lower by pulsatile administration (5.2 +/- 0.8 mU/min) than by continuous infusion (9.2 +/- 1.8 mU/min, p = less than 0.05). Our study demonstrates that pulsed administration of oxytocin every 8 minutes is as effective and safe as continuous intravenous infusion of oxytocin for induction of labor, requires less oxytocin with therefore, a wider margin of safety and is consistent with the pulsatile release of oxytocin during normal labor.