The adverse effects of vitrification on mouse embryo development and metabolic phenotype in offspring.

Embryo vitrification is a standard procedure in assisted reproductive technology. Previous studies have shown that frozen embryo transfer is associated with an elevated risk of adverse maternal and neonatal outcomes. This study aimed to explore the effects of mouse blastocyst vitrification on the phenotype of vitrified-warmed blastocysts, their intrauterine and postnatal development, and the long-term metabolic health of the derived offspring. The vitrified-warmed blastocysts (IVF + VT group) exhibited reduced mitochondrial activity, increased apoptotic levels, and decreased cell numbers when compared to the fresh blastocysts (IVF group). Implantation rates, live pup rates, and crown-rump length at E18.5 were not different between the two groups. However, there was a significant decrease in fetal weight and fetal/placental weight ratio in the IVF + VT group. Furthermore, the offspring of the IVF + VT group at an age of 36 weeks had reduced whole energy consumption, impaired glucose and lipid metabolism when compared with the IVF group. Notably, RNA-seq results unveiled disturbed hepatic gene expression in the offspring from vitrified-warmed blastocysts. This study revealed the short-term negative impacts of vitrification on embryo and fetal development and the long-term influence on glucose and lipid metabolism that persist from the prenatal stage into adulthood in mice.

Association between ambient temperature exposure and pregnancy outcomes in patients undergoing in vitro fertilization in Shanghai, China: a retrospective cohort study.

STUDY QUESTION: Does ambient temperature exposure affect outcomes including clinical pregnancy and live birth in women undergoing IVF? SUMMARY ANSWER: Both extreme cold and hot ambient temperatures were significantly associated with adverse pregnancy outcomes of IVF cycles. WHAT IS KNOWN ALREADY: Heat exposure has been linked to adverse pregnancy outcomes worldwide. However, the effect of ambient temperature on infertile women undergoing IVF treatment is unclear. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study was conducted from a database of 3452 infertile women who underwent their first fresh or frozen embryo transfer in the Shanghai First Maternity and Infant Hospital from April 2016 to December 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: Daily mean ambient temperature exposure for each patient was obtained based on their residential address. Temperature-stratified multiple logistic regression analysis was performed to investigate associations between temperature exposure and pregnancy outcomes after controlling for confounders. Vulnerable sub-groups were identified using forest plots. MAIN RESULTS AND THE ROLE OF CHANCE: The clinical pregnancy rate and live birth rate were 45.7% and 37.1%, respectively. Regarding clinical pregnancy, a higher temperature during cold weather was significantly associated with a higher pregnancy rate in the period about 11 weeks before ovarian stimulation (adjusted odds ratio (aOR) = 1.102, 95% CI: 1.012-1.201). Regarding live birth, an increased temperature during cold weather was significantly related to a higher live birth rate in the period after confirmation of clinical pregnancy or biochemical pregnancy, with the aORs of 6.299 (95% CI: 3.949-10.047) or 10.486 (95% CI: 5.609-19.620), respectively. However, a higher temperature during hot weather was negatively associated with the live birth rate in the periods after confirmation of clinical pregnancy or biochemical pregnancy, with the aORs at 0.186 (95% CI: 0.121-0.285) or 0.302 (95% CI: 0.224-0.406), respectively. Moreover, the decline in live birth rates during cold and hot weather was accompanied by increased rates of early miscarriage (P < 0.05). Stratified analyses identified susceptibility characteristics among the participants. LIMITATIONS, REASONS FOR CAUTION: Climate monitoring data were used to represent individual temperature exposure levels according to the patient's residential address in the study. We were not able to obtain information of personal outdoor activity and use of indoor air conditioners in this retrospective study, which may affect actual temperature exposure. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights that the ambient temperature exposure should be taken into account during IVF treatment and afterwards. There is a need to be alert to extremes in cold and hot ambient temperatures, especially during the period of follicle development and pregnancy. With this knowledge, clinicians can scientifically determine the timing of IVF treatment and reinforce patients' awareness of self-protection to minimize adverse pregnancy outcomes associated with extreme temperatures. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a grant from the Clinical Research Plan of Shanghai Hospital Development Center [SHDC2020CR4080], a grant from the Science and Technology Commission of Shanghai Municipality [19411960500], and two grants from the National Natural Science Foundation of China [81871213, 81671468]. B.W.M. is supported by a NHMRC Investigator grant (GNT1176437). B.W.M. reports consultancy for ObsEva, and research grants from Merck KGaA, Ferring and Guerbet. The other authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Chromatin Accessibility Analysis from Fresh and Cryopreserved Human Ovarian Follicles.

Understanding how gene regulatory elements influence ovarian follicle development has important implications in clinically relevant settings. This includes understanding decreased fertility with age and understanding the short-lived graft function commonly observed after ovarian tissue cryopreservation and subsequent autologous transplantation as a fertility preservation treatment. The Assay for Transposase Accessible Chromatin by sequencing (ATAC-seq) is a powerful tool to identify distal and proximal regulatory elements important for activity-dependent gene regulation and hormonal and environmental responses such as those involved in germ cell maturation and human fertility. Original ATAC protocols were optimized for fresh cells, a major barrier to implementing this technique for clinical tissue samples which are more often than not frozen and stored. While recent advances have improved data obtained from stored samples, this technique has yet to be applied to human ovarian follicles, perhaps due to the difficulty in isolating follicles in sufficient quantities from stored clinical samples. Further, it remains unknown whether the process of cryopreservation affects the quality of the data obtained from ovarian follicles. Here, we generate ATAC-seq data sets from matched fresh and cryopreserved human ovarian follicles. We find that data obtained from cryopreserved samples are of reduced quality but consistent with data obtained from fresh samples, suggesting that the act of cryopreservation does not significantly affect biological interpretation of chromatin accessibility data. Our study encourages the use of this method to uncover the role of chromatin regulation in a number of clinical settings with the ultimate goal of improving fertility.

Potential roles of experimental reproductive technologies in infertile women with diminished ovarian reserve.

In assisted reproductive technology treatment, diminished ovarian reserve (DOR) is a condition of utmost clinical and scientific relevance because of its negative influence on patient outcomes. The current methods of infertility treatment may be unsuitable for many women with DOR, which support the need for development of additional approaches to achieve fertility restoration. Various techniques have been tried to improve the quality and increase the quantity of oocytes in DOR patients, including mitochondrial transfer, activation of primordial follicles, in vitro culture of follicles, and regeneration of oocytes from various stem cells. Herein, we review the science behind these experimental reproductive technologies and their potential use to date in clinical studies for infertility treatment in women with DOR.

Visualization of Balbiani Body disassembly during human primordial follicle activation.

Dormant human oocytes contain a perinuclear super-organelle, called the Balbiani Body, which is not present in mature oocytes. Here, we use confocal imaging to visualize two Balbiani Body markers-mitochondria and the DEAD-box helicase DDX4-in preantral follicles isolated from a 20-year-old female patient. In primordial follicles, mitochondria were concentrated in a ring near the oocyte nucleus, while DDX4 formed adjacent micron-scale spherical condensates. In primary and secondary follicles, the mitochondria were dispersed throughout the oocyte cytoplasm, and large DDX4 condensates were not visible. Our data suggest that the Balbiani Body breaks down during the primordial to primary follicle transition, thus releasing mitochondria and soluble DDX4 protein into the oocyte cytoplasm.

Clinical outcomes after fresh versus frozen embryo transfer in women with advanced reproductive age undergoing in vitro fertilization: a propensity score-matched cohort study.

This retrospective cohort study aimed to compare clinical outcomes following fresh or frozen embryo transfer (FET) in women with advanced reproductive age (ARA). Women aged 35-45 years who underwent their first autologous fresh or frozen cleavage stage embryo transfer cycle in the Centre for Assisted Reproduction of Shanghai First Maternity and Infant Hospital between January 2016 and December 2020 were included. The primary outcome was live birth after the first embryo transfer of the in vitro fertilization (IVF) cycle. Multiple covariates were used for propensity score matching (PSM) and generalized estimating equations were performed to examine the independent association between FET and live birth. Of the total 1453 patients, 327 patients had FET and 1126 patients had fresh ET. After the PSM procedure, 274 patients were included in each group. The live birth rate was 24.8% in the FET group and 25.2% in the fresh ET group (OR 0.98, 95% CI: 0.67-1.44, P = 0.92). Other pregnancy, perinatal and neonatal outcomes were all comparable between the two groups. This study showed that FET did not improve live birth and other clinical outcomes as compared with fresh embryo transfer in women with ARA who underwent their first IVF cycle.

Dissecting the spermatogonial stem cell niche using spatial transcriptomics.

Spermatogonial stem cells (SSCs) in the testis support the lifelong production of sperm. SSCs reside within specialized microenvironments called "niches," which are essential for SSC self-renewal and differentiation. However, our understanding of the molecular and cellular interactions between SSCs and niches remains incomplete. Here, we combine spatial transcriptomics, computational analyses, and functional assays to systematically dissect the molecular, cellular, and spatial composition of SSC niches. This allows us to spatially map the ligand-receptor (LR) interaction landscape in both mouse and human testes. Our data demonstrate that pleiotrophin regulates mouse SSC functions through syndecan receptors. We also identify ephrin-A1 as a potential niche factor that influences human SSC functions. Furthermore, we show that the spatial re-distribution of inflammation-related LR interactions underlies diabetes-induced testicular injury. Together, our study demonstrates a systems approach to dissect the complex organization of the stem cell microenvironment in health and disease.

Sperm DNA damage: how relevant is it clinically?

PURPOSE OF REVIEW: The true impact of the current sperm DNA fragmentation testing needs further scrutiny to assess whether clinically meaningful information is conveyed. RECENT FINDINGS: Various studies have suggested different or no threshold values with assorted tests for the percentage of DNA fragmentation in the ejaculated sperm above which natural conception, fertilization or embryo development and/or clinical pregnancy rates are compromised. Current DNA fragmentation assessment methods provide very little specific information on the nature and severity of the DNA damage detected. Although sperm DNA fragmentation is associated with lower pregnancy rates through natural conception or intrauterine insemination, it does not seem to affect intracytoplasmic sperm injection outcome. Although animal studies demonstrated adverse reproductive effects of sperm DNA fragmentation, any conclusive evidence in humans is yet to be demonstrated. It is not clear whether interventions aimed at enrichment of sperm with decreased DNA fragmentation are effective in preventing the potential adverse effects of sperm DNA fragmentation in humans. Major concern about the use of sperm DNA integrity tests as prognostic parameters is that the direct evaluation of DNA fragmentation in individual sperm fertilizing the oocyte is not possible. SUMMARY: The lack of consensus in defining a clinically relevant standard DNA fragmentation test with a meaningful cut-off level brings challenges in implementing the routine use of sperm DNA integrity assessment in daily practice.

Sperm Cryopreservation Outcomes in Transgender Adolescents Compared with Adolescents Receiving Gonadotoxic Therapy.

Purpose: The target population for fertility preservation recently has been expanded from adolescents with cancer undergoing gonadotoxic chemotherapy to include transgender youth before initiating gender-affirming hormone therapy. Patients and providers may have knowledge deficits regarding options for fertility preservation, accessibility, and feasibility of its techniques, and impact of treatment on future fertility. This study describes outcomes of sperm cryopreservation in transgender male-to-female (affirmed female) youth and compares semen parameters with adolescents diagnosed with cancer. Methods: Medical records of transgender-affirmed female adolescents and adolescent males diagnosed with cancer who underwent sperm cryopreservation at the Fertility and Advanced Reproductive Medicine clinic of the University of Texas (UT) Southwestern Medical Center between March 2015 and March 2020 were reviewed. Demographic data were recorded and values for sperm parameters (volume, count, total count, motility (%), total motile) were collected. When available, hormone levels (luteinizing hormone, follicle-stimulating hormone, testosterone, and estradiol) and Tanner stages were also assessed. The two populations were compared using chi-square analysis and two-sample student's t-test. Data are presented as mean±standard deviation. Results: While semen quality parameters trended lower in transgender youth compared with adolescents with cancer, there was no statistically significant difference between groups. While four out of 18 patients in the transgender group had azoospermia, mean semen quality parameters fell within normal adult reference ranges for both groups. Conclusion: Sperm cryopreservation for transgender youth and adolescents with cancer is feasible, inexpensive, and does not result in significant treatment delays. This information can improve counseling and access to these procedures, particularly in the transgender population.

Biochemistry, molecular biology and cell biology of gonadotropin-releasing hormone antagonists.

PURPOSE OF REVIEW: Gonadotropin-releasing hormone (GnRH) receptors are not only detected in the central nervous system but also in tissues such as ovary, endometrium, breast, gastrointestinal system, placenta and malignant tumors of ovary and breast. The direct role of GnRH-antagonists in ovarian function, implantation, cancer pathogenesis and treatment is under extensive investigation. This study reviews the biochemistry and molecular and cellular biology of GnRH-antagonists as well as GnRH types and their receptors. RECENT FINDINGS: The best clinical evidence with GnRH-antagonists has accumulated in controlled ovarian hyperstimulation protocols for prevention of premature luteinizing hormone surge (cetrorelix, ganirelix) and in the treatment of advanced-stage prostate cancer (abarelix and degarelix). GnRH-GnRH receptor pathways may have a role in the embryo implantation. The controversy still exists whether GnRH antagonist protocols result in slightly decreased clinical pregnancy rates compared with the GnRH agonist protocols. GnRH-antagonists could be used in the near future to treat some cancer types that express GnRH receptors. SUMMARY: GnRH-antagonists have various clinical applications in gynecology, reproductive medicine, urology and oncology. The emergence of well tolerated, orally active GnRH-antagonists may provide an alternative to long-term injections and is likely to have a major impact on the utility of GnRH analogues in the treatment of human diseases.

Specialist physicians' referral behavior regarding preimplantation genetic testing for single-gene disorders: Is there room to grow?

OBJECTIVE: To assess whether primary care specialists' demographics, specialty, and knowledge of preimplantation genetic testing for monogenic disorders (PGT-M) influence their practice patterns. DESIGN: Cross-sectional survey study. SETTING: Academic medical center. PATIENTS: Not applicable. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Objective PGT-M knowledge, subjective comfort with PGT-related topics, PGT care practices (discussions/referrals), and PGT-M implementation barriers. RESULTS: Our survey had 145 respondents: 65 obstetrician/gynecologists, 36 internists, and 44 pediatricians. Overall, 88% believed that patients at a risk of passing on genetic disorders should be provided PGT-M information. However, few discussed PGT-M with their patients (24%) or referred them for testing (23%). Over half (63%) believed that the lack of physician knowledge was a barrier to PGT use. In terms of subjective comfort with PGT, only 1 in 5 physicians felt familiar enough with the topic to answer patient questions. There were higher odds of discussing (odds ratio, 3.21; 95% confidence interval, 1.75-5.87) or referring for PGT (odds ratio, 2.52; 95% confidence interval, 1.41-4.51) for each additional 0.5 correct answers to PGT knowledge-related questions. The odds of referring patients for PGT-M were the highest among obstetrician/gynecologists compared with those among the internists and pediatricians. CONCLUSIONS: Physician specialty and PGT knowledge were associated with PGT-M care delivery practices. Although most specialists believed in equipping at-risk patients with PGT-M information, <1 in 4 discussed or referred patients for PGT. The low levels of PGT-related care among providers may be owed to inadequate knowledge of and comfort with the topic. An opportunity to promote greater understanding of PGT-M among primary care specialists exists and can in turn improve the use of referrals to PGT-M services.

Effects of Early Cumulus Cell Removal on Treatment Outcomes in Patients Undergoing In Vitro Fertilization: A Retrospective Cohort Study.

Background: Early cumulus cell removal combined with early rescue intracytoplasmic sperm injection (ICSI) has been widely practiced in many in vitro fertilization (IVF) centers in China in order to avoid total fertilization failure. However, uncertainty remains whether the pregnancy and neonatal outcomes are associated with early cumulus cell removal. Objectives: To investigate if early cumulus cell removal alone after 4 hours co-incubation of gametes (4 h group), has detrimental effect on the pregnancy and neonatal outcomes in patients undergoing IVF, through a comparison with conventional cumulus cell removal after 20 hours of insemination (20 h group). Methods: This retrospective cohort study included 1784 patients who underwent their first fresh cleavage stage embryo transfer at the Centre for Assisted Reproduction of Shanghai First Maternity and Infant Hospital from June 2016 to December 2018 (4 h group, n=570; 20 h group, n=1214). A logistic regression analysis was performed to examine the independent association between early cumulus cell removal and pregnancy outcomes after adjustment for potential confounders. The neonatal outcomes between the two groups were compared. Results: When compared with the 20 h group, the 4 h group had similar pregnancy outcomes, including rates for biochemical pregnancy, clinical pregnancy, ongoing pregnancy, miscarriage, ectopic pregnancy, multiple pregnancy, live birth. There were 1073 infants delivered after embryo transfer (4 h group, n=337; 20 h group, n=736). Outcomes in both groups were similar for both singleton and twin gestations, including preterm birth rate and very preterm birth rate, mean birth weight, mean gestational age, sex ratio at birth and rate of congenital birth defects. In addition, findings pertaining to singleton gestations were also similar in the two groups for Z-scores (gestational age- and sex-adjusted birth weight), rates of small for gestational age, very small for gestational age, large for gestational age and very large for gestational age infants. Conclusions: In this study early cumulus cell removal alone was not associated with adverse pregnancy and neonatal outcomes. From this perspective, early cumulus cell removal to assess for a potential early rescue ICSI is therefore considered to be a safe option in patients undergoing IVF.

Single-dose administration of a short-acting gonadotropin-releasing hormone agonist does not affect cycle outcome in frozen-thawed embryo transfer cycles.

OBJECTIVE: This prospective study aimed to assess the effect of short-acting gonadotropin-releasing hormone agonist (GnRHa) administration on pregnancy outcomes in frozen-thawed embryo transfer (FET) cycles. METHODS: Patients who planned to have FET in Peking Union Medical College Hospital (China) were recruited for this study and randomly assigned into two groups. Patients in the experimental group (n = 460) received triptorelin acetate on the day of embryo transfer along with routine luteal support. Patients in the control group (n = 433) only received luteal support. One dose (0.1 mg) of a short-acting GnRHa was administered on the day of blastocyte transfer. The rates for clinical pregnancy, biochemical pregnancy, implantation, miscarriage, and ectopic pregnancy were compared between the groups. RESULTS: There were no significant differences in the number and quality of blastocytes transferred between the two groups. In the experimental and control groups, the clinical pregnancy rate was 56.3% and 50.58%, the biochemical pregnancy rate was 15.78% and 18.94%, and the median implantation rate was 39.98% and 38.01%, respectively, with no significant difference between the groups. Biochemical pregnancy and abortion and the ectopic pregnancy rate were not significantly different between the two groups. CONCLUSION: In FET cycles, a GnRHa does not affect the pregnancy outcome.

Association between ambient air pollution and pregnancy outcomes in patients undergoing in vitro fertilization in Shanghai, China: A retrospective cohort study.

BACKGROUND: The effects of ambient air pollutants on adverse pregnancy outcomes have been reported. However, studies about air pollutants exposure and pregnancy outcomes in patients undergoing IVF were limited and inconclusive. To date Shanghai has been the only city in China to implement a compulsory single embryo transfer policy for all patients undergoing their first embryo transfer procedure effective from January 2019. We aimed to investigate the associations between exposure to ambient air pollutants and biochemical pregnancy and live births, and to identify potential vulnerability characteristics of patients undergoing IVF in Shanghai, China. METHODS: A retrospective cohort study was conducted on 2766 infertile patients aged ≤ 45 years who underwent first fresh or frozen-thawed cleavage stage embryo transfer in the Shanghai First Maternity and Infant Hospital during April 2016 and December 2019. Daily average ambient levels of six air pollutants (PM2.5, PM10, NO2, SO2, CO and O3 max-8h) were obtained from fixed air monitors located in closest proximity to patients' residences. The cumulative average level was calculated during three different exposure periods (period1: three months before oocyte retrieval to serum hCG test; period 2: from serum hCG test to live birth outcome; period 3: from three months before oocyte retrieval to live birth). Multiple logistic regression model was performed to investigate associations between exposure to ambient air pollutants and pregnancy outcomes. Stratified analyses were conducted to explore the potential effects modifier. RESULTS: The biochemical pregnancy rate and live birth rate were 54.2% and 36.4%, respectively. The ambient NO2 exposure was significantly associated with a 14% lower pregnancy rate during period 1 (aOR = 0.86, 95%CI: 0.75-0.99). The ambient PM10 was related to significantly increased risk of lowering live birth rate among the patients during period 3 [aOR = 0.88(0.79-0.99)]. Stratified analysis showed that ambient PM10 was also significantly associated with a reduced pregnancy rate (aOR = 0.82, 95% CI: 0.69-0.97) in patients who underwent single embryo transfer during period 1. Subjects who underwent single embryo transfer also had a decreased likelihood of a live birth when exposed to ambient SO2 and O3 during period 3 [aOR = 0.74(0.57-0.95), and 0.92 (0.83-0.98), respectively]. Moreover, O3 exposure was associated with decreased live birth rates in patients living in non-urban areas. Sensitivity analyses indicated robust negative association between PM10 exposure and live birth outcomes. CONCLUSIONS: Our study suggested that exposure to ambient air pollutants, in particular NO2 and PM10, was associated with an increased risk of lower rates of pregnancy and live birth respectively in patients undergoing IVF. Stratified analyses indicated that ambient SO2 and O3 levels were related to adverse pregnancy outcomes in some subgroups of IVF patients in this study. Notably, patients who underwent single embryo transfer were more susceptible to ambient air pollution exposure. Thus, prospective cohort studies are needed to investigate the underlying mechanisms and the susceptibility windows for women undergoing IVF treatment.

Controlled Ovarian Stimulation Protocols Alter Endometrial Histomorphology and Gene Expression Profiles.

Although it is well appreciated that ovarian stimulation protocols for in vitro fertilization (IVF) alter endometrial receptivity, the precise cellular mechanisms are not known. To gain insights into potential mechanisms by which different ovarian stimulation protocols alter the endometrium, we compared histologic and gene expression profiles of endometrium from women undergoing conventional ovarian stimulation for IVF (C-IVF) with those undergoing minimal stimulation with clomiphene citrate (MS-IVF). Sixteen women undergoing MS-IVF (n = 8) or C-IVF (n = 8) were recruited for endometrial biopsy at the time of oocyte retrieval. Endometrial glands were large, tortuous, and secretory with C-IVF but small and undifferentiated with MS-IVF. Whereas RNA sequencing did not reveal changes in estrogen receptor or its co-regulators or classic proliferation associated genes in MS-IVF, together with immunohistochemistry, Wnt signaling was disrupted in endometrium from MS-IVF cycles with significant upregulation of Wnt inhibitors. Secreted frizzled-related protein 1 (sFRP1) was increased fourfold (p < 0.01), and sFRP4 was upregulated sixfold (p < 0.01) relative to C-IVF. Further these proteins were localized to subepithelial endometrial stroma. These data indicate that MS-IVF protocols with CC do not seem to impact endometrial estrogen signaling as much as would be expected from the reported antiestrogenic properties of CC. Rather, the findings of this study highlight Wnt signaling as a major factor for endometrial development during IVF cycles.

Does assisted reproductive technology cause birth defects?

PURPOSE OF REVIEW: Observational studies have reported small but increased risk of birth defects associated with assisted reproductive technology (ART) pregnancies. We intend to review the current data on this issue to provide essential information for patient counseling. RECENT FINDINGS: There is lack of consensus on whether ART per se increases the risk of birth defects, genetic and imprinting disorders, mostly due to the bias inherent to the observational studies, which suggest 30-40% increased risk of birth defects with ART. Recent reports suggest that apparent risks for adverse outcome following ART may be largely related to parental infertility-linked factors. Criteria used to define birth defects bring challenges as well. SUMMARY: All couples undergoing these procedures should be counseled about the current information suggesting elevated risks of birth defects, genetic abnormalities and imprinting disorders associated with infertility and the infertility treatment with ART. In order to elucidate the controversy whether ART is associated with adverse outcome in the offspring, a multinational collaborative effort is needed, as the required sample size is between 10 000 and 84 000 or more.

Effect of single-dose ibuprofen on follicular fluid levels of interleukins in poor responders undergoing in vitro fertilization.

Premature follicular rupture during in vitro fertilization (IVF) is a well-known culprit for cycle cancellation. We sought to evaluate whether a single oral dose of ibuprofen will have an effect on the follicular fluid (FF) levels of inflammatory markers involved in ovulation. This is a prospective within-subjects study following nine patients undergoing IVF. Every patient underwent a first cycle of minimal stimulation IVF followed by a second cycle using the same stimulation protocol, except one oral dose of ibuprofen 800 mg was administered 15-18 h post-trigger injection. FF was obtained during oocyte retrievals of both cycles and analyzed for levels of selected inflammatory markers. A total of 27 cytokines and 9 matrix metalloproteinases (MMPs) were tested. Results demonstrate significantly decreased levels of interleukin (IL)-6, IL-8, granulocyte-colony stimulating factor (G-CSF), eotaxin, MMP3, MMP7, MMP12, and MMP13 in FF of cycles where ibuprofen was administered. However, other cytokines levels, such IL-1 and vascular endothelial growth factor (VEGF), were similar with or without ibuprofen. Levels of MMPs described to be involved in ovulation, namely MMP-2 and MMP-9, were either undetectable or unchanged by ibuprofen, respectively. In conclusion, our data show that one dose of ibuprofen administered orally the day after trigger injection revealed a significant impact on the FF inflammatory milieu. Abbreviations: IVF: in vitro fertilization; MMP: matrix metalloproteinase; IL: interleukins; FF: follicular fluid; VEGF: vascular endothelial growth factor; NSAIDS: non-steroidal anti-inflammatories; POR: poor ovarian response; AMH: anti-Mullerian hormone; TAFC: total antral follicle count; HMG: human menopausal gonadotropin; hCG: human chorionic gonadotropin; COX: cyclooxygenase enzymes; PGH2: prostaglandin H2; RANTES: regulated on activation, normal T expressed and secreted; NF-κb: nuclear factor kappa-light-chain-enhancer of activated B cells.

Effect of Body Weight on Metabolic Hormones and Fatty Acid Metabolism in Follicular Fluid of Women Undergoing In Vitro Fertilization: A Pilot Study.

INTRODUCTION: Obesity is an epidemic affecting more than one-third of adults in the United States. Obese women experience decreased fertility, explained in part by oocyte quality. Since follicular fluid (FF) provides an important microenvironment for oocyte growth, we sought to evaluate the effect of increased body weight on FF levels of 11 metabolic hormones and fatty acid metabolism. METHODS: The FF was collected from 25 women (10 normal weight, 10 overweight, and 5 obese) with diminished ovarian reserve undergoing in vitro fertilization (IVF) following a minimal stimulation protocol. Hormone levels were determined by multiplex immunoassay using the MAGPIX (Luminex, Austin, Texas) instrument. Fatty acid metabolites were determined using gas and ultra-high pressure liquid chromatography coupled with mass spectrometry. RESULTS: Levels of hormones related to glucose and energy homeostasis and regulation of fat stores (insulin, glucagon, glucagon-like peptide-1, C-peptide, and leptin) were increased significantly in FF from obese women compared to FF from nonobese(normal weight and overweight) women. Interestingly, FF levels of branched-chain amino acids (BCAA) isoleucine, leucine, and valine as well as uric acid, isocaproic acid, butanoic acid, tyrosine, threonine, glycine, and methionine correlated positively with body mass index. CONCLUSION: This pilot study demonstrates significant alterations in the FF milieu of obese women undergoing IVF, which may contribute to the decreased fecundity of obese women. Although the impact of this environment on oocyte and embryo development is not fully realized, these changes may also lead to imprinting at the genomic level and long-term sequelae on offspring.

Inflammatory status influences aromatase and steroid receptor expression in endometriosis.

Aberrant up-regulation of aromatase in eutopic endometrium and implants from women with endometriosis has been reported. Aromatase induction may be mediated by increased cyclooxygenase-2 (COX-2). Recently, we demonstrated that progesterone receptor (PR)-A and PR-B serve an antiinflammatory role in the uterus by antagonizing nuclear factor kappaB activation and COX-2 expression. PR-C, which antagonizes PR-B, is up-regulated by inflammation. Although estrogen receptor alpha (ERalpha) is implicated in endometriosis, an antiinflammatory role of ERbeta has been suggested. We examined stage-specific expression of aromatase, COX-2, ER, and PR isoform expression in eutopic endometrium, implants, peritoneum, and endometrioma samples from endometriosis patients. Endometrial and peritoneal biopsies were obtained from unaffected women and those with fibroids. Aromatase expression in eutopic endometrium from endometriosis patients was significantly increased compared with controls. Aromatase expression in endometriosis implants was markedly increased compared with eutopic endometrium. Aromatase mRNA levels were increased significantly in red implants relative to black implants and endometrioma cyst capsule. Moreover, COX-2 expression was increased in implants and in eutopic endometrium of women with endometriosis as compared with control endometrium. As observed for aromatase mRNA, the highest levels of COX-2 mRNA were found in red implants. The ratio of ERbeta/ERalpha mRNA was significantly elevated in endometriomas compared with endometriosis implants and eutopic endometrium. Expression of PR-C mRNA relative to PR-A and PR-B mRNA was significantly increased in endometriomas compared with eutopic and control endometrium. PR-A protein was barely detectable in endometriomas. Thus, whereas PR-C may enhance disease progression, up-regulation of ERbeta may play an antiinflammatory and opposing role.

Androstenedione up-regulation of endometrial aromatase expression via local conversion to estrogen: potential relevance to the pathogenesis of endometriosis.

CONTEXT: Up-regulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity may enhance their survival via local estrogen synthesis, which may lead to endometriosis. The factors that mediate induction of aromatase in the endometrium are not well defined, but increased expression of steroidogenic factor (SF)-1 may play a role. OBJECTIVE: The objective of the study was to determine whether androstenedione (A4), the predominant sex steroid in peritoneal fluid, regulates endometrial aromatase expression. DESIGN: This was a cell/tissue culture study. SETTING: The study was conducted at an academic center. METHODS: Quantitative real-time PCR, HPLC, and chromatin immunoprecipitation were used in this study. RESULTS: Treatment of cultured human endometrial explants and stromal cells with A4 (10 nm) significantly up-regulated expression of aromatase mRNA transcripts containing exon IIa at their 5'-ends. In endometrial stromal cells and the human endometrial surface epithelial (HES) cell line, induction of aromatase mRNA by A4 was associated with increased expression of SF-1. In HES cells, tritiated A4 was metabolized to estradiol, testosterone (T), dihydrotestosterone, and androstanediol. Both estradiol and T, but not nonaromatizable androgens, up-regulated aromatase and SF-1 mRNA in HES cells. Chromatin immunoprecipitation revealed that A4 enhanced recruitment of SF-1 to its response element (-136 bp) upstream of CYP19 exon IIa. This, together with the findings that both estrogen receptor antagonist, ICI 182,780, and aromatase inhibitor, fadrozole, suppressed A4 and T induction of aromatase and SF-1 mRNA, indicates that the inductive effects of A4 and T are mediated by their conversion to estrogens. CONCLUSIONS: Exposure of endometrial cells to A4 may enhance CYP19 gene expression through its aromatization to estrogens.