Comparison of RNA content from hydrophobic interaction chromatography-isolated seminal plasma exosomes from intrauterine insemination (IUI) pregnancies.

Male factors account for roughly half of infertility cases, with most male infertility diagnosed as idiopathic. Researchers predicting intrauterine insemination success rates have identified multiple prognostic factors, including semen parameters and seminal fluid composition. Seminal plasma contains extracellular exosomes, which contain RNAs and proteins involved in spermatogenesis. The contents of seminal plasma exosomes may be an indicator of overall sperm quality or fertility potential; therefore, analysis of exosomes may provide a measure for sperm viability and fertilization potential. In our study, exosomes were isolated and purified from seminal plasma obtained from IUI treatments with known pregnancy outcomes. We used a unique method to isolate the exosomes which made use of the hydrophobic interaction chromatography method. RNASeq was performed on RNAs from the purified exosomes. This analysis revealed holistic trends, including increased expression associated with RNA originating from chromosomes 1, 10, 12, 16 and 21. Overall, total RNA was significantly decreased and rRNA was significantly increased in successful IUI attempts. Furthermore, we found specific mRNAs and lincRNAs associated with positive versus negative pregnancy outcomes. Our study isolated and purified seminal plasma exosomes without ultracentrifugation, and it provides further evidence for differences in seminal plasma exosome molecular contents associated with pregnancy status.

Apoptotic qPCR gene expression array analysis demonstrates proof-of-concept for rapid blastocoel fluid-conditioned media molecular prediction.

PURPOSE: Successful identification of transcriptomic biomarkers within human IVF embryos may enhance implantation prediction and provide insights not available through conventional embryo biopsy genomic analysis. We demonstrate proof-of-concept for a methodology to assess overall embryo gene expression using qPCR with blastocoel fluid-conditioned media by examining the comparative presence of apoptotic genes. METHODS: Blastocoel fluid-conditioned media were collected from 19 embryos (11 euploid) following trophectoderm biopsy of day-5 ICSI-IVF blastocysts. Media were assessed for apoptotic gene expression via qPCR. Statistical analysis of gene expression was conducted via Wilcoxon Signed-Ranks test (overall expression), multivariate ANOVA (functional gene groups), and chi-square test of independence (gene level). RESULTS: A significantly higher overall apoptotic gene expression within euploid versus aneuploid embryos (p = 0.001) was observed. There was significantly (p = 0.045) higher expression of pro-apoptotic genes between implanted and not implanted embryos. Pro- vs. anti-apoptotic gene expression from all euploid embryos approached significance (p = 0.053). The ploidy status-based claim is further substantiated at the gene level with significantly higher expression of BBC3 (p = 0.012) and BCL2L13 (p = 0.003) in euploid embryos compared to aneuploid embryos. CONCLUSIONS: In this preliminary study, we demonstrate that (1) qualitative analysis of blastocoel fluid-conditioned media gene expression is possible, (2) global trends of expression are potentially related to clinical outcomes, and (3) gene-level expression trends exist and may be another viable metric for comparative expression between samples. The presence of statistical significance within analyses conducted with this sample size warrants a larger investigation of blastocoel fluid-conditioned media as an additional beneficial predictive tool for future IVF cases.

The expression of pregnancy-associated plasma protein-A (PAPP-A) in human blastocoel fluid-conditioned media: a proof of concept study.

PURPOSE: The aim of this study was to determine if pregnancy-associated plasma protein-A (PAPP-A), typically measured in maternal serum and a potential predictor of adverse maternal and fetal outcomes such as spontaneous miscarriage, pre-eclampsia, and stillbirth, is expressed in blastocoel fluid-conditioned media (BFCM) at the embryonic blastocyst stage. DESIGN: This is an in vitro study. METHODS: BFCM samples from trophectoderm-tested euploid blastocysts (n = 80) from in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) patients were analyzed for PAPP-A mRNA. BFCM was obtained from blastocyst stage embryos in 20 uL drops. Blastocysts underwent trophectoderm biopsy for preimplantation genetic testing for aneuploidy prior to blastocyst vitrification and BFCM collection for snap freezing. cfDNA was synthesized using BFCM collected from 80 individual euploid blastocysts. Next, real-time qPCR was performed to detect expression of PAPP-A with GAPDH for normalization of expression in each sample. RESULTS: PAPP-A mRNA was detected in 45 of 80 BFCM samples (56.3%), with varying levels of expression across samples. CONCLUSION: Our study demonstrates the expression of PAPP-A in BFCM. To our knowledge, this is the first study to report detection of PAPP-A mRNA in BFCM. Further studies are required and underway to investigate a greater number of BFCM samples as well as the possible correlation of PAPP-A expression with pregnancy outcomes of transferred euploid blastocysts. If found to predict IVF and obstetric outcomes, PAPP-A may provide additional information along with embryonic euploidy for the selection of the optimal blastocyst for embryo transfer.

Comparative analysis of viral infection outcomes in human seminal fluid from prior viral epidemics and Sars-CoV-2 may offer trends for viral sexual transmissibility and long-term reproductive health implications.

BACKGROUND: Viral detection in seminal fluid indicates their potential for both sexual transmission and impairment of reproductive health. Review of the mechanistic entry, sexual transmission and viral impacts for patients during major recent viral outbreaks of Zika virus (ZIKV), Ebola virus (EBOV), severe acute respiratory syndrome (SARS)-coronavirus (CoV), and SARS-coronavirus 2 (CoV-2) (the virus which causes COVID-19) provides a framework to discuss this potential. AIM: Comparative analysis of prior viral presence on seminal fluid against current (preliminary) findings for SARS-CoV-2 to predict biological implications of the novel coronavirus upon current sexual transmissibility, viral presence, and reproductive health. METHODOLOGY AND FINDINGS: Literature review was conducted using PubMed and Google Scholar databases. ZIKV and EBOV were found to be present in semen and to be sexually transmitted, leading the World Health Organization (WHO) to update their guidelines on prevention of the two viruses to include refraining from sexual contact. There are conflicting studies regarding the presence of SARS-CoV in male reproductive tissue, but it has been linked to testicular atrophy and orchitis. To date, two studies have detected SARS-CoV-2 RNA in semen, while seven studies have reported no positive detection. CONCLUSIONS: Though unlikely in the majority of cases, SARS-CoV-2 can potentially be present in seminal fluid, although there are no reports of sexual transmission to date. Prior epidemics raise significant concerns regarding the long-term reproductive health capacity for patients who are affected by entry of Sars-CoV-2 into the reproductive tract, therefore more study is needed to clarify the impacts to reproductive health.

This review describes the detection of viruses in seminal fluid and their sexual transmission, focusing on the major viral outbreaks of Zika virus (ZIKV), Ebola virus (EBOV), severe acute respiratory syndrome (SARS)-coronavirus (CoV), and SARS-coronavirus 2 (CoV-2). ZIKV and EBOV were found to be present in semen and to be sexually transmitted, leading the World Health Organization (WHO) to update their guidelines on prevention of the two viruses to include refraining from sexual contact. There are conflicting studies regarding the presence of SARS-CoV in male reproductive tissue, but it has been linked to testicular atrophy and orchitis. To date, two studies have detected SARS-CoV-2 RNA in semen, while seven studies have reported no positive detection. More studies must be completed to accurately determine its risk of sexual transmission to ensure mitigation of further transmission and understand the long-term implications of SARS-CoV-2 on the reproductive health of recovered patients.

Relationship between blastocoel cell-free DNA and day-5 blastocyst morphology.

PURPOSE: Cell-free DNA (cfDNA) which is present in the blastocoel cavity of embryos is believed to result from physiological apoptosis during development. This study assessed cfDNA content and caspase-3 protease activity in day-5 IVF blastocysts to determine if there was a correlation with embryo morphology. METHODS: Day-5 IVF blastocysts were scored according to the Gardner and Schoolcraft system (modified to generate a numerical value) and cfDNA was collected following laser-induced blastocoel collapsing prior to cryopreservation in 25 µL of media. cfDNA was quantified via fluorospectrometry and apoptotic activity was assessed via a caspase-3 protease assay using a fluorescent peptide substrate. Data were compared by linear regression. RESULTS: A total of 32 embryos were evaluated. There was a significant (p < 0.01) and positive correlation (cfDNA = 104.753 + (11.281 × score); R2 = 0.200) between embryo score and cfDNA content. A significant (p < 0.05) and positive correlation (cfDNA = 115.9 + (0.05 × caspase-3); R2= 0.128) was observed between caspase-3 activity and cfDNA levels. There was no significant relationship between caspase-3 activity and embryo morphology score. CONCLUSIONS: This study provides further evidence that cfDNA is present in blastocoel fluid, can be quantified, and positively correlates with embryonic morphology. There is also evidence that at least a portion of the cfDNA present is from intracellular contents of embryonic cells that underwent apoptosis. Additional studies are warranted to determine other physiological sources of the cfDNA in blastocyst fluid and to determine the relationship with cfDNA content, embryo morphology, and chromosomal ploidy status plus implantation potential.

Presence of anti-müllerian hormone in the squirrel monkey (Saimiri boliviensis): gender and seasonal differences.

BACKGROUND: Anti-mullerian hormone (AMH) is a glycoprotein of the transforming growth factor-ß superfamily secreted by male Sertoli cells and female ovarian granulosa cells. This study determined squirrel monkey AMH levels as influenced by gender and seasonality. METHODS: Squirrel monkey sera AMH were measured by an enzymatically amplified two-site immunoassay. RESULTS: A significant difference (P<0.001) was found in AMH levels between male (mean=3.46 ng/ml) and female squirrel monkeys (mean=22.12 ng/ml). A significant difference (P<0.05) was found in male AMH levels between breeding (mean=4.21 ng/ml) and non-breeding seasons (mean=2.78 ng/ml). No significant differences were found between female groups. CONCLUSIONS: Anti-mullerian hormone levels in female squirrel monkeys are the highest in any primate species reported, whereas in the male, levels are within reported ranges. The AMH assay may allow us soon to assess the squirrel monkey fertility potential as a function of various factors.

Clinical Utilities of Anti-Müllerian Hormone.

The anti-Müllerian hormone (AMH) plays an essential role in sex determination in early embryonic development. Through a series of sequential steps that follows inheriting an XY chromosome, Sertoli cell differentiation upregulates the expression of AMH-suppressing Müllerian duct development and maintains the AMH at a high level until puberty. In females, the AMH is produced by granulosa cells of follicles beginning in the second half of fetal life and continues through adulthood, with a steady decline through the reproductive years and severe decline at menopause, until levels eventually become undetectable. The AMH is essential for the regulation of follicular maturation via the recruitment of primordial follicles throughout folliculogenesis. AMH serum concentration in women strongly correlates with ovarian reserve quantity and reflects ovulation potential. Because the AMH is expressed almost exclusively by growing follicles before FSH-dependent selection, it commonly serves as a marker for ovarian function in various clinical situations, including in the diagnosis and pathogenesis of polycystic ovarian syndrome, artificial reproductive technology, and predictions of menopause or premature ovarian failure.

Fluorescent-dependent comparative Ct method for qPCR gene expression analysis in IVF clinical pre-implantation embryonic testing.

The use of quantitative PCR (qPCR) and other polymerase chain reaction (PCR)-based methods in the field of human in vitro fertilization blastocoel fluid analysis can potentially be utilized for assisting clinicians in embryo selection based on specific gene expression patterns. Since typical Comparative cycle threshold (Ct ) analysis utilizes one threshold for runs per gene target and requires an inherent control group, this method is inadequate for analysis of small stochastic systems, such as embryonic-derived fluid. We mathematically demonstrate analytical modifications upon the Comparative Ct qPCR workflow to incorporate a variable fluorescence threshold (utilizing only the parameters defined in the Comparative Ct method), and subsequently demonstrate the typical workflow in which this modified method can successfully quantifiably analyze embryonic blastocoel fluid qPCR analysis.

Patient engagement in fertility research: bench research, ethics, and social justice.

BACKGROUND: Patient and Public Involvement (PPI) in research is increasingly being utilized to better connect patients and researchers. The Patient Engagement Studio (PES) supports PPI in research by working directly with researchers throughout various stages of their projects. Recently, two researchers presented to the PES for assistance with their project, Embryo+™. The purpose of Embryo+™ is to decrease miscarriage rates using RNA sequencing technology that screens for the most viable embryos. To date, no examples of PPI directly in the planning or implementation of bench research concerning in vitro fertilization and embryo transfer have been identified. MAIN BODY: Embryo+™ researchers met in-person with the PES two times (fall 2019; each meeting had 9 PES members in attendance) for initial feedback and protocol development. After these meetings, PES leadership and Embryo+™ researchers decided that the unique nature of the project merited a PPI evaluation. Subsequent evaluation of engagement efforts occurred by reviewing the PES reports for the Embryo+™ researchers, conducting two recorded web-based discussion meetings with the PES (summer 2020; meeting 1 n = 7; meeting 2 n = 6), and a brief survey (n = 13). The discussion meetings provided an opportunity for the PES members to define engagement themes through consensus via verbal agreement to the studio director's periodic summaries during the discussions. Combining survey results and PES themes allowed for a broad discussion for meaningful engagement. The Embryo+™ researchers established trust with the patients by changing some of their language in response to patient suggestions, allowing for unintended ethical conversations, and implementing the patient developed protocols. Overall, the patient experts thought this project was very meaningful and valuable, quantified by a mean loyalty score 89.43 (s.d. 10.29). CONCLUSION: Bench science researchers may need additional PPI training prior to engaging with patient groups. PPI in this project was successful in large part due to this training, where the director emphasized the importance of gaining trust with the patients. The researchers applied what they learned and several examples of how to develop trust with patients are discussed. If trust is established, PPI in an ethically charged, basic science research study can be both valuable and successful.

Patients are increasingly becoming involved in all stages of the research process. Patient and public involvement has been shown to answer questions that matter to patients, increase enrollment in studies, and improve the spread of research results to the public. However, there are limited evaluations of patient engagement in basic science research (research performed in a laboratory setting in various fields). Here, we provide an example of patients effectively involved in the planning and implementation of an ethically complex study in the field of Assistive Reproductive Technology. A Patient Engagement Studio, affiliated with an academic health center, directly connects patients and researchers through bi-monthly meetings. Recently, two researchers presented their project, Embryo+™, to the studio during the planning stage of their study. This project aims to use a new testing technology to reduce miscarriage rates. The researchers presented to the studio twice (fall 2019), and two follow-up meetings were conducted with the patients (summer 2020). Also, the patients completed a survey evaluating how engaged they felt with the project. Through the meetings, the researchers changed their language in response to patient feedback, and patients developed project protocols. Survey results showed that the patients thought this project was very meaningful and valuable. Overall, this evaluation shows that patients can add value to contentious bench science projects, particularly in the field of Assistive Reproductive Technology.

Embryo Biopsy Can Offer More Information Than Just Ploidy Status.

As a byproduct of increasing infertility cases, the use of medically assisted reproduction (MAR) has increased. As such, the need to gain information regarding the implantation potential of specific MAR preimplantation embryos prior to transfer has become increasingly critical. One potential source of this information is contained in the blastocoel fluid from day 5/6 embryos. This fluid contains cell-free DNA, proteins, RNA, metabolites, exosomes, etc., and analysis of these contents provides clinicians with an opportunity to gain more data regarding potential of each embryo. While application of preimplantation genetic testing for aneuploidies (PGT-A) may be limited to women of advanced maternal age or with recurrent pregnancy loss, the fluid taken at the time of embryo biopsy can be analyzed for any frozen embryo as well as PGT-A embryos. In both cases, blastocoel fluid analysis provides information regarding a preimplantation embryo's potential for implantation. Moreover, as remnants of apoptosis, embryonic cell-free DNA (cfDNA) and mRNA may lead clinicians to better understand and predict the extent of self-correction occurring within the preimplantation embryo. While analysis of blastocoel components are not yet viable replacements for PGT-A, their study may still reveal critical clinical information about the implantation potential for any given embryo.

High seminal platelet-activating factor acetylhydrolase activity in men with spinal cord injury.

Spinal cord injury (SCI) causes male infertility, with low sperm motility the major long-term cause. It has been suggested in previous studies that some seminal components may be responsible for the pathological asthenozoospermia. It is hypothesized that platelet-activating factor (PAF) acetylhydrolase (PAFah), which originates in the epididymis and other accessory sexual glands, may be a causative factor. This enzyme catalyzes PAF to acetate and biologically inactive lyso-PAF. PAF is well recognized to be an important phospholipid mediator that stimulates sperm motility and enhances sperm capacitation and fertilization. The present study was designed to analyze differences in PAFah activity in semen of men with SCI and age-matched healthy men. PAFah assay reagent kits were used to measure enzymatic activity by monitoring the production rates of 4-nitrophenol on a spectrophotometer during a given interval. The results showed that subjects with SCI had a higher concentration of PAFah than men in the control group (P < .001). A statistically significant negative correlation was found between enzymatic activity and sperm motility (r(2) = 0.8449; P < .001). Further studies will determine whether seminal vesicle dysfunction in men with SCI leads to abnormal PAFah activity, resulting in low sperm motility.

Impact of body mass index values on sperm quantity and quality.

Body mass index (BMI) has been demonstrated to affect female fertility; however, little information is available on the impact of BMI on male fertility or semen parameters. Therefore, the study objective was to determine the relationship between BMI and semen parameters, including sperm chromatin integrity. We analyzed data on semen samples from 520 men who were grouped based upon calculated BMI values (normal, 20-24 kg/m(2); overweight, 25-30 kg/m(2); obese, >30 kg/m(2)). The data collected included patient height and weight, semen volume, sperm concentration, percent sperm motility, percent sperm morphology (normal forms), and sperm chromatin integrity (DNA fragmentation index [DFI]). Data were analyzed by regression analysis and analysis of variance (ANOVA) with Tukey's test for multiple pairwise comparisons. The overall BMI mean (+/-SEM) was 27.5 (+/-0.49) kg/m(2). Linear regression revealed a significant (P < .05) and negative relationship between BMI and the total number of normal-motile sperm cells. ANOVA revealed a significant difference (P < .05) in the total number of normal-motile sperm cells among the different BMI groups. The number of normal-motile sperm cells per BMI group was as follows: normal, 18.6 x 10(6); overweight, 3.6 x 10(6); and obese, (0.7) x 10(6). All multiple pairwise comparisons were found to be significantly (P < .05) different. The overall DFI mean (+/-SEM) was 24.7 (+/-2.57). Linear regression revealed a significant (P < .05) and positive relation between BMI and DFI. Men presenting with a BMI greater than 25 kg/m(2) have fewer chromatin-intact normal-motile sperm cells per ejaculate. Therefore, to ensure maximum fertility potential, patients may be advised to reduce body weight.

Inhibin B is lower and anti-Müllerian hormone is similar in serum of men with spinal cord injuries compared to controls.

Men with spinal cord injury have a unique semen profile characterized by normal sperm concentration but abnormally low sperm motility and viability. The purpose of our study was to determine if new diagnostic information could be obtained for this population by measuring serum concentrations of inhibin B and anti-Müllerian hormone. These hormones, as well as follicle stimulating hormone, luteinizing hormone, and testosterone, were measured in 30 men with spinal cord injury and 15 age-matched control subjects. Serum concentrations of inhibin B and testosterone were significantly lower in the spinal cord injury group compared to the control group. A statistically significant negative relationship was observed between serum concentrations of inhibin B and follicle stimulating hormone in both the spinal cord injury group and the control group, and between inhibin B and luteinizing hormone in the spinal cord injury group only. A significant positive relationship was also observed between inhibin B and sperm concentration in the spinal cord injury group. Although serum concentrations of inhibin B were significantly lower in the spinal cord injury group than in controls, inhibin B and anti-Müllerian hormone serum concentrations did not provide an additional diagnostic tool for male infertility in this population. This is the first study to determine serum concentrations of inhibin B and anti-Müllerian hormone in men with spinal cord injury.

Platelet-activating factor significantly enhances intrauterine insemination pregnancy rates in non-male factor infertility.

OBJECTIVE: To determine the efficacy of treating semen specimens with platelet-activating factor (PAF) before IUI. DESIGN: Prospective randomized double-blinded study of PAF treatment of sperm for patients with a history of infertility undergoing IUI. SETTING: Private infertility center. INTERVENTION(S): Patients had ovulation induction therapy with clomiphene citrate (CC) or gonadotropin, two IUIs per month with PAF treatment. MAIN OUTCOME MEASURE(S): Clinical pregnancy rates. RESULT(S): There was a significant difference in IUI pregnancy rates per cycle between control (10/56; 17.9%) and PAF (14/47; 29.8%) treatment groups in the normal male study arm. There was a significant difference in cumulative IUI pregnancy rates between control (10/35; 28.6%) and PAF (14/26; 53.9%) patient groups in the normal male study arm. There was no significant difference in IUI pregnancy rates per cycle between control (12/124; 9.7%) and PAF (14/119; 11.8%) treatment groups in the male factor study arm. There was no significant difference in cumulative IUI pregnancy rates between control (12/46; 26.1%) and PAF (14/38; 36.8%) patient groups in the male factor study arm. There was a significant difference in overall cumulative IUI pregnancy rates between control (21/81; 25.9%) and PAF (27/64; 42.2%) patient groups. CONCLUSION(S): The inclusion of PAF into the IUI sperm wash procedure significantly improves pregnancy rates. However, the significant improvement can only be shown to affect men presenting with normal semen parameters.

The pig platelet-activating factor receptor gene is expressed at the mRNA level in different tissues and is mapped to chromosome 6.

After the pig platelet-activating factor receptor (PAFr) gene was cloned and sequenced, the chromosomal location of this gene was studied using a pig/rodent somatic cell hybrid panel containing 27 cell lines. The results indicated that the pig PAFr gene is located on SSC6q22-23. Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is thought to be very important in the animal reproductive processes. Its function is mediated through a membrane-bound receptor. Pig PAFr mRNA distribution in different tissues was tested using reverse transcription and PCR (RT-PCR) reactions. All tissues examined expressed PAFr. Using a pig PAFr gene DNA competitor, PAFr expression was quantificated. The pig PAFr mRNA expression level was estimated to be from 1 x 10(2) to 1.2 x 10(4) copies of complementary DNA (cDNA) per 50 ng of total RNA. The highest level was found in lung, and the lowest in the skeletal muscle. These results demonstrated that PAFr was differentially expressed in pig tissues.

Decreased inhibin B responses following recombinant human chorionic gonadotropin administration in normal women and women with polycystic ovary syndrome.

OBJECTIVE: To determine whether granulosa cells contribute to excess androgen production, by assessing inhibin B (Inh B) responses to hCG in women with polycystic ovary syndrome (PCOS) and in normal women. DESIGN: Prospective study. SETTING: Academic medical center. PATIENT(S): Twenty women with PCOS and 16 normal women. INTERVENTION(S): Blood samples obtained before and 24 hours after injection of 25 µg recombinant hCG (r-hCG). MAIN OUTCOME MEASURE(S): Basal and stimulated Inh B, E2, androstenedione (A), and T responses after r-hCG administration. RESULT(S): In normal and PCOS women, r-hCG induced a significant reduction of Inh B levels. Lowered Inh B responses were not related to body mass index, PCOS status, or age by multivariate regression. Recombinant hCG significantly increased serum A and E2 in both normal and PCOS women. CONCLUSION(S): In normal and PCOS women, Inh B production was decreased following r-hCG administration. These findings strongly suggest that in PCOS women androgen excess is not enhanced by LH-stimulated Inh B production. CLINICAL TRIAL REGISTRATION NUMBER: NCT00747617.

Enhancement of sperm motility using pentoxifylline and platelet-activating factor.

Enhancement of sperm motility can effectively improve assisted reproductive technique outcomes. Here we describe two (pentoxifylline and platelet-activating factor) popular sperm motility enhancers and their respective methods.

Development of a second generation anti-Müllerian hormone (AMH) ELISA.

AMH is a glycoprotein dimer composed of two 72kDa monomers linked by disulfide bridges. It belongs to the transforming growth factor-ß family. AMH performs various physiological functions. In males, AMH is secreted by the Sertoli cells of the testis. During embryonic development, AMH is responsible for Müllerian duct regression. AMH continues to be produced by the testicles until puberty and then decreases slowly to residual post-puberty values. In females, AMH is produced in small amounts by ovarian granulosa cells after birth, until menopause, and then becomes undetectable. A two-step, sandwich-type enzymatic microplate assay has been developed to measure AMH levels in 20 µL of sample in less than 3h. AMH calibrators range from 0.2 to 28 ng/mL. The antibodies used in the assay bind to the mature region of AMH, which is more stable to proteolysis compared to prohormone region. The AMH Gen II assay (Beckman Coulter, Inc., Webster, Texas) was standardized to the Immunotech (IOT, Beckman Coulter, Inc., Marseilles, France) AMH assay. AMH Gen II, when compared to IOT using 120 serum samples in the range of 0-20.4 ng/mL yielded a correlation coefficient of 0.98 and a slope of 1.0. Total imprecision, calculated on four samples over 40 runs, four replicates per run, between two lots using CLSI EP5-A guidelines, was 5.7% at 3.8 ng/mL, 7.7% at 4.4 ng/mL, 5.8% at 14 ng/mL and 5.3% at 16.4 ng/mL. The average analytical sensitivity calculated by the interpolation of the mean plus two standard deviations of 16 replicates of the zero calibrator on two independent lots was 0.08ng/mL. Dilution and spiking studies showed an average recovery of 91-110%. Lot-to-lot comparison of two independent lots testing 38 serum samples (1.5-33 ng/mL range) yielded a slope of 1.01, intercept of -0.08 ng/mL and r of 0.99. When potential interferents (hemoglobin, triglycerides, and bilirubin) were added at two times the physiological concentrations, AMH concentrations were within ± 10% of the control. A highly specific and reproducible microplate AMH Gen II assay has been developed to standardize the measurement of AMH between methods. The performance of the AMH Gen II assay is ideal for investigation into the physiologic roles of AMH in men and women.