Anti-Müllerian hormone is a predictor of medium-term cumulative live birth following in vitro fertilization/intracytoplasmic sperm injection: A retrospective study.

OBJECTIVES: This study aims to examine the capacity of anti-Müllerian hormone (AMH) to predict cumulative live birth rate (CLBR) following IVF/ICSI within 36 months since start of treatment. STUDY DESIGN: This is a cohort study of women seeking IVF/ICSI fertility treatment in a private Australian IVF clinic in a single calendar year. Live births were monitored over three years following start date of IVF/ICSI. The impact of serum AMH level on the CLBR was assessed using Cox's proportional hazard models, and its incremental values in the prediction of CLBR were evaluated. RESULTS: The CLBRs were significantly higher in women with AMH levels in the highest (>44.5 pmol/L; 87.0%, 95% CI 79.2% - 95.1%) and in the middle two quartiles (between 11.5 and 44.5 pmol/L; 81.0%, 95% CI 74.2% - 87.6%), compared with AMH levels below the 25th percentile (≤11.5 pmol/L; 63.2%, 95% CI 53.2% - 74.5%). Approximately half of the women with AMH in the lowest quartile conceived a live birth within 12 months of starting IVF compared with two-thirds of the women in the upper three quartiles. After adjusting for confounders, AMH remained a significant, albeit slight predictor of CLBR with a fall of 3 pmol/L equating to an 1% decrease in CLBR. The AMH's added values into the prediction of live birth were slight, indicated by a net reclassification improvement of 13.8%. The value is lower than that of maternal age (35.1%). CONCLUSIONS: Serum AMH level was a significant slight predictor of CLBR following IVF/ICSI. AMH should not be used to exclude women from IVF/ICSI however, women with low AMH should be counselled on the likelihood of taking longer to achieve a live birth than individuals with normal AMH levels.

The predictive value of anti-Müllerian hormone for natural conception leading to live birth in subfertile couples.

RESEARCH QUESTION: What is the predictive value of serum anti-Müllerian hormone (AMH) level for natural conception and its clinical effect on subfertile couples? DESIGN: A retrospective cohort of ovulatory women seeking fertility consultation in a private fertility clinic. Couples who had an immediate indication for IVF were excluded. All natural conceptions leading to live birth before the start of assisted reproductive technology were followed within 12 months of the initial consultation. A prediction model was developed by updating the Hunault model with serum AMH to predict the probabilities of achieving a natural conception leading to live birth. RESULTS: A total of 325 couples were included in the final analysis. The estimated cumulative proability of achieving natural conception leading to live birth within 12 months was 20.9% (95% CI 12.9% to 28.2%). The categorical net reclassification improvement of AMH is 7.6%. For couples with a predicted chance of natural conception changed from poor (<30%) by the reference model to good (≥30%) by the updated model, the cumulative natural conception rate leading to live birth was 52.0%. For couples who had predicted chance of natural conception changed from good to poor by the updated model, the rate was 18.9%. CONCLUSIONS: The addition of serum AMH to the routine fertility work-up may improve prognosis-based treatment policy and help to prevent unnecessary costs and stress for couples. Prospective validation of the updated model with AMH is required before clinical application.

Effect of Intralipid infusion on peripheral blood T cells and plasma cytokines in women undergoing assisted reproduction treatment.

OBJECTIVES: Intravenous infusion of Intralipid is an adjunct therapy in assisted reproduction treatment (ART) when immune-associated infertility is suspected. Here, we evaluated the effect of Intralipid infusion on regulatory T cells (Treg cells), effector T cells and plasma cytokines in peripheral blood of women undertaking IVF. METHODS: This prospective, observational pilot study assessed Intralipid infusion in 14 women exhibiting recurrent implantation failure, a clinical sign of immune-associated infertility. Peripheral blood was collected immediately prior to and 7 days after intravenous administration of Intralipid. Plasma cytokines were measured by Luminex, and T-cell subsets were analysed by flow cytometry. RESULTS: A small increase in conventional CD8+ T cells occurred after Intralipid infusion, but no change was seen in CD4+ Treg cells, or naïve, memory or effector memory T cells. Proliferation marker Ki67, transcription factors Tbet and RORγt, and markers of suppressive capacity CTLA4 and HLA-DR were unchanged. Dimensionality-reduction analysis using the tSNE algorithm confirmed no phenotype shift within Treg cells or other T cells. Intralipid infusion increased plasma CCL2, CCL3, CXCL8, GM-CSF, G-CSF, IL-6, IL-21, TNF and VEGF. CONCLUSION: Intralipid infusion elicited elevated pro-inflammatory cytokines, and a minor increase in CD8+ T cells, but no change in pro-tolerogenic Treg cells. Notwithstanding the limitation of no placebo control, the results do not support Intralipid as a candidate intervention to attenuate the Treg cell response in women undergoing ART. Future placebo-controlled studies are needed to confirm the potential efficacy and clinical significance of Intralipid in attenuating cytokine induction and circulating CD8+ T cells.

Obesity related metabolic endotoxemia is associated with oxidative stress and impaired sperm DNA integrity.

BACKGROUND: Obesity is known to be associated with inflammation, oxidative stress and a resulting reduction in sperm DNA integrity. Importantly, obesity is also reported to be associated with an increase in intestinal permeability with the passage of intestinal bacteria into the circulation (metabolic endotoxemia) that triggers a systemic state of inflammation and resultant oxidative stress. Therefore, we hypothesised that this obesity related increase in intestinal permeability and resultant metabolic endotoxemia (ME) may activate inflammation within the male reproductive tract, leading to increased reactive oxygen species production, sperm oxidative stress and a decline in DNA integrity. RESULTS: Our pilot study of 37 infertile men confirmed a significant positive correlation between body mass index (BMI), increased intestinal permeability (serum zonulin), metabolic endotoxaemia (LBP), sperm DNA oxidative damage (seminal 8 OhDG) and increasing levels of sperm DNA fragmentation (Halosperm). Metabolic endotoxemia was positively correlated with increasing levels of sperm DNA oxidative damage with this relationship remaining significant, even after adjustment for relevant confounders such as age, BMI and days of abstinence. These observations suggest that metabolic endotoxemia and its associated oxidative stress may be a key driver of sperm DNA damage in obese men. CONCLUSION: This study confirms a link between obesity, increasing intestinal permeability and endotoxin exposure, and oxidative mediated sperm DNA damage. This warrants further investigation to fully understand the effect of metabolic endotoxemia on male reproductive function which could result in the new therapies to improve male fertility potential.

Interferon-gamma inhibits seminal plasma induction of colony-stimulating factor 2 in mouse and human reproductive tract epithelial cells.

Seminal fluid interacts with the female reproductive tract to initiate a permissive immune response that facilitates embryo implantation and pregnancy success. The immune-regulatory cytokine interferon-γ (IFNG), which can be elevated in seminal plasma, is associated with reduced fertility. Here, we investigated how IFNG influences the female immune response to seminal fluid. In human Ect1 cervical epithelial cells, IFNG added at physiologically relevant concentrations substantially impaired seminal plasma-induced synthesis of key cytokines colony-stimulating factor 2 (CSF2) and interleukin-6 (IL6). Seminal fluid-induced CSF2 synthesis was also suppressed in the uterus of mice in vivo, when IFNG was delivered transcervically 12 h after mating. Transforming growth factor B1 (TGFB1) is the major seminal fluid signaling factor which elicits CSF2 induction, and IFNG exhibited potent dose-dependent suppression of CSF2 synthesis induced by TGFB1 in murine uterine epithelial cells in vitro. Similarly, IFNG suppressed TGFB1-mediated CSF2 induction in Ect1 cells and human primary cervical epithelial cells; however, IL6 regulation by IFNG was independent of TGFB1. Quantitative PCR confirmed that CSF2 regulation by IFNG in Ect1 cells occurs at the gene transcription level, secondary to IFNG suppression of TGFBR2 encoding TGFB receptor 2. Conversely, TGFB1 suppressed IFNG receptor 1 and 2 genes IFNGR1 and IFNGR2. These data identify IFNG as a potent inhibitor of the TGFB-mediated seminal fluid interaction with relevant reproductive tract epithelia in mice and human. These findings raise the prospect that IFNG in the male partner's seminal fluid impairs immune adaptation for pregnancy following coitus in women.

Seminal plasma pro-inflammatory cytokines interferon-γ (IFNG) and C-X-C motif chemokine ligand 8 (CXCL8) fluctuate over time within men.

STUDY QUESTION: Do seminal plasma pro-inflammatory cytokines interferon-γ (IFNG) and C-X-C motif chemokine ligand 8 (CXCL8) vary within individual men over time? SUMMARY ANSWER: IFNG exhibits substantial variation that is independent of duration of abstinence but correlates with lipopolysaccharide (LPS) content, while CXCL8 varies moderately in association with duration of abstinence. WHAT IS KNOWN ALREADY: Pro-inflammatory cytokines IFNG and CXCL8 in seminal fluid can adversely impact male and female fertility. Other cytokines as well as sperm parameters fluctuate considerably within individuals over time, but whether IFNG and CXCL8 vary similarly, and the determinants of variance, are unknown. STUDY DESIGN, SIZE, DURATION: Between two and seven semen samples were collected from 14 proven fertile donors at 6-10 week intervals over the course of ~12 months, to assess variation over time in cytokines and LPS, and to investigate relationships with sperm parameters and possible regulatory factors. PARTICIPANTS/MATERIALS, SETTING, METHODS: The concentrations and total amounts per ejaculate of IFNG and CXCL8 were determined using commercial ELISA. Sperm parameters were assessed according to World Health Organization (WHO) IV standards and LPS was measured by limulus amebocyte lysate (LAL) assay. Mixed model analysis was utilized to determine the relative contribution of between- and within-individual factors in explaining variance. Relationships between cytokines, LPS and sperm parameters, as well as effect of age and duration of abstinence, were investigated by correlation analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Within-individual variability contributed to total variance particularly for both IFNG, CXCL8 and LPS, and was a stronger determinant than between-individual variability for IFNG and LPS. Normal sperm motility correlated inversely with CXCL8, and sperm concentration correlated inversely with LPS. Duration of abstinence was a determinant of total CXCL8, but not IFNG or LPS. Associations between LPS, IFNG and CXCL8 suggest IFNG and perhaps CXCL8 are influenced by microbial populations. LIMITATIONS, REASONS FOR CAUTION: A limited number of donors from a single clinic were investigated. Clinical information on complete microbiology, BMI, nutrition, smoking and other lifestyle factors was unavailable. Further studies are required to determine whether the findings can be generalized to larger populations and different ethnicities. WIDER IMPLICATIONS OF THE FINDINGS: These data reveal substantial variation over time in pro-inflammatory seminal fluid cytokines and imply existence of microbial or other environmental regulatory factors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the National Health and Medical Research Council of Australia. The authors have no competing interests to disclose.

Seminal plasma transforming growth factor-ß, activin A and follistatin fluctuate within men over time.

STUDY QUESTION: Do seminal plasma transforming growth factor-ß (TGFB) cytokines vary within individuals over time, and does this relate to sperm parameters, age or prior abstinence? SUMMARY ANSWER: Activin A and follistatin, and to a lesser extent TGFB1, TGFB2 and TGFB3, vary within individuals over time, in association with duration of abstinence. WHAT IS ALREADY KNOWN: Seminal plasma TGFB cytokines can influence sperm function and reproductive success through interactions with the female reproductive tract after coitus. Over time, individual sperm parameters fluctuate considerably. Whether seminal fluid TGFB cytokines vary similarly, and the determinants of any variance, is unknown. STUDY DESIGN, SIZE, DURATION: Between two and seven semen samples were collected from each of 14 fertile donors at 6-10 week intervals over the course of 12 months, then seminal plasma cytokines and sperm parameters were measured. PARTICIPANTS/MATERIALS, SETTING AND METHOD: The concentrations and total amounts per ejaculate of TGFB1, TGFB2, TGFB3, activin A and follistatin were determined using commercial assays. Sperm parameters were assessed according to WHO IV standards. Mixed model analysis was utilised to determine the relative contribution of between- and within-individual factors to the variance. Relationships between cytokines and sperm parameters, as well as effect of age and duration of abstinence, were investigated by correlation analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Within-individual variability contributed to the total variance for all cytokines and sperm parameters, and was a stronger determinant than between-individual variability for activin A and follistatin as well as for total sperm concentration and sperm motility. Positive correlations between each of the three TGFB isoforms, and activin and follistatin, suggest co-regulation of synthesis. Duration of abstinence influenced total content of TGFB1, TGFB2, activin A and follistatin. TGFB1 correlated inversely with age. LIMITATIONS, REASONS FOR CAUTION: A limited number of donors from a single clinic were investigated. Clinical information on BMI, nutrition, smoking and other lifestyle factors was unavailable. Further studies are required to determine whether the findings can be generalised to larger populations and different ethnicities. WIDER IMPLICATIONS OF THE FINDINGS: These data reveal substantial variation over time in seminal fluid cytokines and indicate that repeated analyses are required to gain precise representative data on an individual's status. Within-individual variation in seminal fluid components should be taken into account when investigating seminal fluid cytokines. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the National Health and Medical Research Council of Australia, ID453556 and APP1041332. The authors have no competing interests to disclose.

Consistent high clinical pregnancy rates and low ovarian hyperstimulation syndrome rates in high-risk patients after GnRH agonist triggering and modified luteal support: a retrospective multicentre study.

STUDY QUESTION: Are clinical pregnancy rates satisfactory and the incidence of OHSS low after GnRH agonist trigger and modified intensive luteal support in patients with a high risk of ovarian hyperstimulation syndrome (OHSS)? SUMMARY ANSWER: GnRH agonist trigger combined with 1500 IU hCG at the time of oocyte retrieval and subsequent estradiol and progesterone replacement in OHSS high-risk patients can facilitate fresh embryo transfer with high clinical pregnancy rates and a low risk of severe OHSS. WHAT IS KNOWN ALREADY: Conventional luteal support packages are inadequate to facilitate a fresh transfer after a GnRH agonist trigger. A low dose of hCG (1500 IU) after oocyte aspiration can be used to replace the actions of early luteal LH to sustain implantation and the function of the early corpus luteum, although the level of risk of severe OHSS with this strategy is unclear. STUDY DESIGN, SIZE, DURATION: This international multicentre retrospective case study, including 275 women at high risk of OHSS, was undertaken during the period January 2011-December 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women were identified as at high risk of OHSS, based on IVF response, ovarian reserve characteristics and previous history of having had treatment, in three clinical IVF centres in UK, Belgium and Australia. All three centres used a GnRH agonist trigger followed by one bolus of 1500 IU hCG 1h after oocyte retrieval. Moreover, the luteal phase was supported with daily vaginal progesterone and twice daily estradiol valerate. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 275 autologous cycles with fresh transfer were undertaken in a cohort of high-risk women as defined by baseline characteristics [median (interquartile range)]: age 31.6 (29-35) years, antral follicle count median 25 (18-34) and anti-Müllerian hormone median 49.1 pmol/l (35.2-69.3). At the end of stimulation, the peak estradiol median of 12 000 pmol/l (9400-15 914) and the mean oocyte yield of 17.8 ± 8.4 confirmed a high response. The overall clinical pregnancy rate was 41.8% per cycle started, with only two cases of severe OHSS reported (0.72%). No significant differences in clinical pregnancy rates between centres were identified. LIMITATIONS, REASONS FOR CAUTION: This is a retrospective study and future randomized controlled trials will be able to compare whether these outcomes can be improved upon by either segmentation of the stimulation cycle and embryo transfer or alternative aggressive luteal support strategies. WIDER IMPLICATIONS OF THE FINDINGS: In women who are undergoing ovarian stimulation and who develop an excessive ovarian response, the use of a GnRH agonist trigger combined with modified luteal support can provide the opportunity to proceed to fresh embryo transfer with adequate clinical pregnancy rates. However, this procedure will not completely eliminate the risk of OHSS and for women with an extreme ovarian response or with significant comorbidity, where the possibility of severe OHSS is unacceptable, we recommend GnRH agonist trigger followed by a freeze-all policy to completely avoid OHSS.

TGF-ß mediates proinflammatory seminal fluid signaling in human cervical epithelial cells.

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-ß1, TGF-ß2, and TGF-ß3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-ß3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-ß isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-ß induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-ß neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-ß isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-ß in the male partner's seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.

Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus.

In mice, seminal fluid elicits an inflammation-like response in the female genital tract that activates immune adaptations to advance the likelihood of conception and pregnancy. In this study, we examined whether similar changes in leukocyte and cytokine parameters occur in the human cervix in response to the male partner's seminal fluid. After a period of abstinence in proven-fertile women, duplicate sets of biopsies were taken from the ectocervix in the periovulatory period and again 48 h later, 12 h after unprotected vaginal coitus, vaginal coitus with use of a condom, or no coitus. A substantial influx of CD45(+) cells mainly comprising CD14(+) macrophages and CD1a(+) dendritic cells expressing CD11a and MHC class II was evident in both the stratified epithelium and deeper stromal tissue after coitus. CD3(+)CD8(+)CD45RO(+) T cells were also abundant and increased after coitus. Leukocyte recruitment did not occur without coitus or with condom-protected coitus. An accompanying increase in CSF2, IL6, IL8, and IL1A expression was detected by quantitative RT-PCR, and microarray analysis showed genes linked with inflammation, immune response, and related pathways are induced by seminal fluid in cervical tissues. We conclude that seminal fluid introduced at intercourse elicits expression of proinflammatory cytokines and chemokines, and a robust recruitment of macrophages, dendritic cells, and memory T cells. The leukocyte and cytokine environment induced in the cervix by seminal fluid appears competent to initiate adaptations in the female immune response that promote fertility. This response is also relevant to transmission of sexually transmitted pathogens and potentially, susceptibility to cervical metaplasia.

The distribution of immune cells and macrophages in the endometrium of women with recurrent reproductive failure. II: adenomyosis and macrophages.

Adenomyosis, a condition usually associated with multiparity, is not generally seen as a cause of infertility. However, recent studies have reported a reduction in IVF implantation rates and a link with miscarriage, suggesting that adenomyosis may interfere with successful implantation. To investigate this hypothesis, the clinical records and laboratory results, which routinely include immunohistochemical examination of a late luteal phase endometrial biopsy for leukocytes, were retrospectively reviewed for 64 women with implantation failure and who previously had been screened for the presence of adenomyosis by pelvic MRI. The presence of either diffuse or "adenomyoma" type of adenomyosis was associated with a marked increase (p=0.004) in the density of macrophages and natural killer cells in the endometrial stroma, compared to those women with mild focal adenomyosis or no disease. These findings point to an immunological mechanism by which adenomyosis might interfere with successful embryo implantation.

Increased gonadotrophin stimulation does not improve IVF outcomes in patients with predicted poor ovarian reserve.

PURPOSE: This retrospective study was carried out to evaluate whether increasing the starting dose of FSH stimulation above the standard dose of 150 IU/day in patients with low predicted ovarian reserve can improve IVF outcomes. METHOD: A total of 122 women aged less than 36 years in their first cycle of IVF were identified as having likely low ovarian reserve based on a serum AMH measurement below 14 pmol/l. Thirty five women were administered the standard dose of 150 IU/day FSH, while the remaining 87 received a higher starting dose (200-300 IU/day FSH). There were no significant differences in age, BMI, antral follicle count, serum AMH, FSH or aetiology of infertility between the two dose groups. RESULTS: No significant improvement in oocyte and embryo yield or pregnancy rates was observed following an upward adjustment of FSH starting dose. While increasing the dose of FSH above 150 IU/day did not produce any adverse events such as OHSS, it did consume an extra 1,100 IU of FSH per IVF cycle. CONCLUSION: The upward FSH dose adjustment in anticipation of low ovarian reserve can not be advocated as it is both expensive and of no proven clinical value.

Anti-Müllerian hormone as a predictor of IVF outcome.

Serum anti-Müllerian hormone (AMH) concentration and antral follicle count (AFC) are two increasingly popular static measures used to predict ovarian reserve prior to IVF treatment. While they have been shown to be good predictors of oocyte yield during ovarian stimulation, their status as indicators of oocyte quality and pregnancy rates is currently uncertain. The present study measured baseline concentrations of serum AMH and FSH, and AFC from 126 women undergoing IVF treatment. These data were then related to IVF outcomes. As expected, patients with lower serum AMH and AFC produced a significantly (P < 0.001) lower number of oocytes compared with patients with higher serum AMH/AFC. Fertilization rates in patients with lower serum AMH were significantly inferior compared with patients with higher serum AMH, irrespective of whether IVF (P = 0.043) or intracytoplasmic sperm injection (P = 0.006) was used to achieve fertilization. These low AMH patients yielded fewer oocytes, had lower fertilization rates, generated fewer embryos, and had a higher incidence of miscarriage during fresh transfers, ultimately culminating in a halving of the pregnancy rate per IVF cycle compared with the high AMH group.

Seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells.

Exposure to semen elicits an inflammatory response in the female reproductive tract of rodents and other animals. The nature and regulation of any similar response in humans is poorly understood. This study investigated seminal plasma induction of inflammatory cytokine and chemokine gene regulation in human cervical and vaginal epithelial cells in vitro. Affymetrix microarray gene profiling revealed that inflammatory cytokine genes were prevalent among 317 known genes differentially expressed in immortalized ectocervical epithelial (Ect1) cells after incubation with pooled human seminal plasma. A dose- and time-dependent induction by seminal plasma of IL8, IL6, CSF2 and CCL2 mRNA expression in Ect1 cells was verified by quantitative RT-PCR. This was accompanied by increases in Ect1 secretion of immunoactive gene products IL-8, IL-6, GM-CSF and MCP-1. Similar cytokine responses were elicited in primary ectocervical epithelial cells. Endocervical epithelial (End1) and vaginal epithelial (Vk2) cells were less responsive to seminal fluid, with induction of IL-8 and MCP-1, but not GM-CSF or IL-6. In a panel of 10 seminal plasma samples, considerable variation in inflammatory cytokine-inducing activity was evident. These experiments show that seminal plasma can elicit expression of a range of inflammatory cytokines and chemokines in reproductive tract epithelia, and implicate the ectocervix as the primary site of responsiveness, with gene-specific differences in the kinetics and site-restrictedness of the response. Seminal factor regulation of inflammatory cytokines in the cervical epithelium is implicated in controlling the immune response to seminal antigens, and defence against infectious agents introduced at intercourse.

Reduced expression of IL-6 and IL-1alpha mRNAs in secretory phase endometrium of women with recurrent miscarriage.

A diverse array of cytokines is implicated in regulating the immune adaptation and endometrial tissue remodelling events that facilitate successful embryo implantation and early placental development. The aim of this study was to evaluate expression of mRNAs encoding a panel of immunoregulatory cytokines in the endometrium of fertile women and women experiencing recurrent miscarriage using highly sensitive, quantitative RT-PCR assays. Endometrial biopsies were collected during the mid-secretory phase of the menstrual cycle from women classified as proven fertile (control; n=12) and women experiencing unexplained recurrent miscarriage (RM; n=9). Reduced IL-6 mRNA and reduced IL-1alpha mRNA were independently associated with recurrent miscarriage. Altered expression was evident after accounting for variation in the composition of endometrial biopsies by normalization of data to epithelial and mesenchymal cell-specific transcripts, cytokeratin-18 mRNA and vimentin mRNA, respectively. The relative abundance of mRNAs encoding LIF, GM-CSF, IFNgamma, IL-1beta, IL-4, IL-5, IL-10, IL-12p40, TNFalpha, TGFbeta1, TGFbeta2 and TGFbeta3 were not altered in recurrent miscarriage tissue. Associations between expression of IL-10, LIF, GM-CSF and TGFbeta2 suggest that regulatory circuits link the transcription of these cytokine genes. Inadequate expression of IL-6 and IL-1alpha mRNAs in endometrial tissue may predispose to recurrent miscarriage through a perturbed maternal immune response, effects on decidual tissue remodeling and angiogenesis, or dysregulated trophoblast differentiation and invasion. Quantitative RT-PCR assays for these cytokines in endometrial biopsies may be a realistic strategy for development of novel diagnostics for predisposition to recurrent miscarriage.

Primary unexplained infertility is associated with reduced expression of the T-regulatory cell transcription factor Foxp3 in endometrial tissue.

A receptive endometrial environment requires adequate immunological tolerance to protect the implanting embryo from maternal immune rejection. Studies in mice implicate CD4+CD25+ T-regulatory (Treg) cells as essential mediators of immune tolerance in pregnancy. The aim of this study was to evaluate the link between Treg cells and fertility in women. Expression of Foxp3, a master regulator of Treg cell differentiation, was quantified in endometrial tissue from women experiencing primary unexplained infertility and normal fertile women. Endometrial biopsies were collected during the mid-secretory phase of the menstrual cycle from women meeting rigorously defined criteria for unexplained infertility after experiencing repeated failed cycles of IVF treatment (infertile, n = 10), or women classified as proven fertile (control, n = 12). Expression of Foxp3 mRNA was reduced approximately two-fold in the tissue of infertile women. In contrast, mRNAs encoding T cell transcription factors T-bet and GATA3, associated with differentiation of Th1 and Th2 CD4+ T cells respectively, were unchanged. Treg cell differentiation is controlled by TGFbeta, but the relative abundance in endometrial tissue of TGFbeta1, TGFbeta2, TGFbeta3 mRNAs was not changed in infertile women. Cytokines influencing Th1 and Th2 cell differentiation, including IFNgamma, IL-2, IL-4, IL-5, IL-10 and IL-12p40, as well as dendritic cell-regulating cytokines IL-1alpha, IL-1beta, IL-6, LIF, GM-CSF and TNFalpha were also expressed similarly regardless of fertility status. The finding of reduced endometrial Foxp3 implicates impaired differentiation of uterine T cells into the Treg phenotype as a key determinant of fertility in women. The factors underpinning this aberration in the immune response remain to be identified.

Singleton births after routine preimplantation genetic diagnosis using exclusion testing (D4S43 and D4S126) for Huntington's disease.

OBJECTIVE: To develop exclusion testing protocols for Huntington's disease (HD) linkage markers suitable for use in a clinical preimplantation genetic diagnosis (PGD) setting for couples in whom a partner was at 50% risk of inheriting HD, but who choose not to undergo presymptomatic mutation testing. DESIGN: Preimplantation genetic diagnosis using exclusion testing. SETTING: In vitro fertilization clinic. PATIENT(S): Three couples with family histories of HD, two couples opposed to direct mutation testing. INTERVENTION(S): Development of single-cell polymerase chain reaction tests for PGD for the HD mutation and two HD gene-flanking markers (D4S43 and D4S126), allowing the identification of an individual embryo as being at either low or high risk for developing HD without being diagnostic of the presence of the mutation. MAIN OUTCOME MEASURE(S): D4S43, D4S126, and HD mutation. RESULT(S): After PGD for HD, couple 1 gave birth to a healthy girl after a frozen embryo transfer, and genetic status was confirmed by prenatal diagnosis to be very low risk for developing HD. Couple 2 gave birth to a healthy boy after their second cycle of PGD, and couple 3, after a third cycle, gave birth to a boy with congenital heart defects, which were successfully corrected with surgery at age 5 days. Both couples 2 and 3 declined prenatal testing, and therefore relinquished the opportunity to confirm the PGD. CONCLUSION(S): Preimplantation genetic diagnosis for HD using exclusion testing resulted in three live singleton births after six oocyte recovery procedures. The diagnostic protocol provided couples the opportunity to minimize the likelihood of disease transmission to their children, without the requirement for predictive testing.

Anti-mullerian hormone as a marker of ovarian reserve.

OBJECTIVE: To analyse the usefulness of plasma anti-mullerian hormone (AMH) measurement as a tool for assessing ovarian reserve in a general infertility population. MATERIALS AND METHODS: Plasma AMH levels were analysed in 238 women aged 18-46 years during day 3-5 of their menstrual cycle. All 238 patients had follicle stimulating hormone (FSH) levels less than 10 i.u./L, suggesting normal ovarian reserve on traditional FSH criteria. Eighty-seven patients gave their consent to correlate their AMH levels with IVF oocyte retrieval outcome. Patients producing > or = 8 oocytes were classified as having normal ovarian reserve, while those producing < or = 4 oocytes were classified as having poor ovarian reserve. RESULTS: Plasma AMH levels remained relatively static (20-25 pmol/L) from 18 to 29 years of age. By 30 years of age, plasma AMH levels start to drop rapidly, reaching only 10 pmol/L by 37 years. Despite this 50% fall in AMH levels between 29 and 37 years of age, minimal changes in FSH levels were observed. Using a cut off value of 8.1 pmol/L, plasma AMH assessment could predict poor ovarian reserve on a subsequent IVF cycle with a sensitivity of 80% and a specificity of 85%. CONCLUSIONS: Plasma AMH assessments are superior to FSH in identifying women with reduced ovarian reserve. Anti-mullerian hormone assessment should be considered as a useful adjunct to FSH/oestradiol levels and antral follicle count when estimating ovarian reserve.

Seminal 'priming' for protection from pre-eclampsia-a unifying hypothesis.

Conventional belief holds that an immune response to ejaculate antigens should interfere with fertilisation and establishment of pregnancy. However, emerging evidence now supports the opposing view-that insemination acts to activate maternal immune mechanisms exerting a positive effect on reproductive events. In a response well documented in rodents, semen triggers an influx of antigen-presenting cells into the female reproductive tract which process and present paternal ejaculate antigens to elicit activation of lymphocytes in the adaptive immune compartment. Transforming growth factor beta (TGFbeta), a cytokine present in abundance in seminal plasma, initiates this inflammatory response by stimulating the synthesis of pro-inflammatory cytokines and chemokines in uterine tissues. Lymphocyte activation is evident in lymph nodes draining the uterus and leads to hypo-responsiveness in T-cells reactive with paternal alloantigens. TGFbeta has potent immune-deviating effects and is likely to be the key agent in skewing the immune response against a Type-1 bias. Prior exposure to semen in the context of TGFbeta can be shown to be associated with enhanced fetal-placental development late in gestation. In this paper, we review the experimental basis for these claims and propose the hypothesis that, in women, the partner-specific protective effect of insemination in pre-eclampsia might be explained by induction of immunological hypo-responsiveness conferring tolerance to histocompatibility antigens present in the ejaculate and shared by the conceptus.