Progestogen-driven B7-H4 contributes to onco-fetal immune tolerance.

Immune tolerance mechanisms are shared in cancer and pregnancy. Through cross-analyzing single-cell RNA-sequencing data from multiple human cancer types and the maternal-fetal interface, we found B7-H4 (VTCN1) is an onco-fetal immune tolerance checkpoint. We showed that genetic deficiency of B7-H4 resulted in immune activation and fetal resorption in allogeneic pregnancy models. Analogously, B7-H4 contributed to MPA/DMBA-induced breast cancer progression, accompanied by CD8+ T cell exhaustion. Female hormone screening revealed that progesterone stimulated B7-H4 expression in placental and breast cancer cells. Mechanistically, progesterone receptor (PR) bound to a newly identified -58 kb enhancer, thereby mediating B7-H4 transcription via the PR-P300-BRD4 axis. PR antagonist or BRD4 degrader potentiated immunotherapy in a murine B7-H4+ breast cancer model. Thus, our work unravels a mechanistic and biological connection of a female sex hormone (progesterone) to onco-fetal immune tolerance via B7-H4 and suggests that the PR-P300-BRD4 axis is targetable for treating B7-H4+ cancer.

Emergency department utilization for ovarian hyperstimulation syndrome.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a rare, but serious, risk of assisted reproductive technologies. In severe cases, patients may present to the emergency department (ED) for assessment, treatment of related complications, and even in-patient admission. Significant effort has been made to reduce the incidence and complications of OHSS; however, it is unknown if these strategies have decreased patient presentation for treatment in the ED. PURPOSE: To assess ED utilization for OHSS over time and to examine admission rates, patient demographics, and charges. METHODS: Retrospective longitudinal study utilizing data from the Nationwide Emergency Department Sample Database and the National ART Surveillance System. All ED visits between 2006 and 2016 with an ICD-9 or -10 diagnosis of OHSS were included. Demographics including age, geographic location, and income quartile and alternative diagnoses, admission rates, overall charges, and number of stimulation cycles annually were assessed. RESULTS: The number of ovarian stimulation cycles steadily increased from 2006 (n = 110,183) to 2016 (n = 157,721), while the number of OHSS-related ED visits remained relatively stable (APC 2.08, p = 0.14). Admission rates for OHSS decreased from 52.7% in 2006 to 33.1% in 2016 (APC -4.43%, p < 0.01). The average charge for OHSS-related ED visits almost doubled from 2006 to 2016 (APC 8.53, p < 0.01) and was significantly higher than charges for non-OHSS-related visits for age-matched controls (p < 0.01). CONCLUSION: Despite an increase in total stimulation cycles, there was no significant change in the estimated number of patients presenting to the ED; however, admission rates significantly declined. These observations suggest a possible shift in the severity and/or management of OHSS during the study period.

Establishing a reproductive biorepository for basic and translational research: experience developing the reproductive subjects registry and sample repository.

PURPOSE: To report experience designing and establishing a reproductive registry and sample biorepository and to describe initial subject characteristics and biospecimens. METHODS: Beginning in December 2017, patients presenting for reproductive care at the University of Michigan were approached for study enrollment. Following consent, subjects completed detailed reproductive and health questionnaires. A variety of reproductive specimens and tissues were collected and processed for multiple downstream applications. RESULTS: Subject enrollment began in December of 2017. There are currently 1798 subjects enrolled. Female participants report a variety of reproductive disorders. Available samples include semen, sperm, follicular fluid, granulosa cells, immature oocytes, ovarian and uterine tissue, and blood samples. CONCLUSION: We report the successful establishment of a reproductive registry and sample biorepository. Furthermore, we describe methods for collection and storage of a variety of reproductive tissue processed for multiple downstream translational applications.

Lack of trusted diagnostic tools for undetermined male infertility.

Semen analysis is the cornerstone of evaluating male infertility, but it is imperfect and insufficient to diagnose male infertility. As a result, about 20% of infertile males have undetermined infertility, a term encompassing male infertility with an unknown underlying cause. Undetermined male infertility includes two categories: (i) idiopathic male infertility-infertile males with abnormal semen analyses with an unknown cause for that abnormality and (ii) unexplained male infertility-males with "normal" semen analyses who are unable to impregnate due to unknown causes. The treatment of males with undetermined infertility is limited due to a lack of understanding the frequency of general sperm defects (e.g., number, motility, shape, viability). Furthermore, there is a lack of trusted, quantitative, and predictive diagnostic tests that look inside the sperm to quantify defects such as DNA damage, RNA abnormalities, centriole dysfunction, or reactive oxygen species to discover the underlying cause. To better treat undetermined male infertility, further research is needed on the frequency of sperm defects and reliable diagnostic tools that assess intracellular sperm components must be developed. The purpose of this review is to uniquely create a paradigm of thought regarding categories of male infertility based on intracellular and extracellular features of semen and sperm, explore the prevalence of the various categories of male factor infertility, call attention to the lack of standardization and universal application of advanced sperm testing techniques beyond semen analysis, and clarify the limitations of standard semen analysis. We also call attention to the variability in definitions and consider the benefits towards undetermined male infertility if these gaps in research are filled.

Secondary Prevention of Intrauterine Adhesions Following Hysteroscopic Surgery in Women With Asherman Syndrome: Is Something Better Than Nothing?

Asherman syndrome is a reproductive disorder characterized by intrauterine adhesions and amenorrhea, infertility, abnormal placentation, or pregnancy loss. Treatment of Asherman syndrome involves hysteroscopic lysis of adhesions. Many surgeons utilize postoperative measures such as hormone therapy, solid mechanical devices, or barrier gels to prevent recurrent adhesions in this setting. However, there is limited high-quality evidence to support their use. Additional research is needed on the safety and efficacy of these commonly used methods to guide patient care.

Histone modification signatures in human sperm distinguish clinical abnormalities.

PURPOSE: Alternations to the paternal epigenome, specifically the components of sperm chromatin, can lead to infertility in humans and potentially transmit aberrant information to the embryo. One key component of sperm chromatin is the post-translational modification of histones (PTMs). We previously identified a comprehensive profile of histone PTMs in normozoospermic sperm; however, only specific histone PTMs have been identified in abnormal sperm by antibody-based approaches and comprehensive changes to histone PTM profiles remain unknown. Here, we investigate if sperm with abnormalities of total motility, progressive motility, and morphology have altered histone PTM profiles compared to normozoospermic sperm samples. METHODS: Discarded semen samples from 31 men with normal or abnormal semen parameters were analyzed for relative abundance of PTMs on histone H3 and H4 by "bottom-up" nano-liquid chromatography-tandem mass spectrometry. RESULTS: Asthenoteratozoospermic samples (abnormal motility, forward progression, and morphology, n = 6) displayed overall decreased H4 acetylation (p = 0.001) as well as alterations in H4K20 (p = 0.003) and H3K9 methylation (p < 0.04) when compared to normozoospermic samples (n = 8). Asthenozoospermic samples (abnormal motility and progression, n = 5) also demonstrated decreased H4 acetylation (p = 0.04) and altered H4K20 (p = 0.005) and H3K9 methylation (p < 0.04). Samples with isolated abnormal progression (n = 6) primarily demonstrated decreased acetylation on H4 (p < 0.02), and teratozoospermic samples (n = 6) appeared similar to normozoospermic samples (n = 8). CONCLUSION: Sperm samples with combined and isolated abnormalities of total motility, progressive motility, and morphology display distinct and altered histone PTM signatures compared to normozoospermic sperm. This provides evidence that alterations in histone PTMs may be important for normal sperm function and fertility.

Medical and elective fertility preservation: impact of removal of the experimental label from oocyte cryopreservation.

PURPOSE: The purpose of this study was to compare baseline characteristics and ovarian stimulation outcomes between patients presenting for medically indicated vs. elective fertility preservation consultation and to determine the impact of the 2013 ASRM guidelines on oocyte cryopreservation on the patient population presenting for fertility preservation consultation. METHODS: Retrospective cohort study conducted at an academic center. Study population included 332 patients presenting for medically indicated fertility preservation consultation and 210 patients presenting for elective consultation. RESULTS: Patients presenting for elective fertility preservation consultation were more likely to be of advanced age, non-Caucasian, highly educated, single, nulligravid, and meet criteria for diminished ovarian reserve (DOR). Additionally, patients presenting electively were more likely to have fertility insurance benefits. A higher percentage of patients with insurance benefits for oocyte cryopreservation proceeded to stimulation. There were no differences in stimulation parameters or number of retrieved oocytes between the groups when adjusted for age. Following release of the ASRM guidelines on oocyte cryopreservation, there was no difference in the percentage of patients in the medical group who proceeded with stimulation; however, a higher percentage of patients presenting electively underwent ovarian stimulation. CONCLUSION: Although the populations presenting for medical compared with elective fertility preservation differ at baseline, ovarian stimulation parameters and outcomes are similar when adjusted for age. Insurance benefits for fertility preservation are not comprehensive and impact the decision to proceed with stimulation in all patients. The publication of the ASRM guidelines on oocyte cryopreservation increased utilization of this technology among patients presenting electively; however, they remained at an advanced age and with decreased ovarian reserve parameters.

The impact of obesity on reproductive health and metabolism in reproductive-age females.

Obesity is a highly prevalent chronic disease that impacts >40% of reproductive-aged females. The pathophysiology of obesity is complex and can be understood simply as a chronic energy imbalance whereby caloric intake exceeds caloric expenditure with an energy surplus stored in adipose tissue. Obesity may be categorized into degrees of severity as well as different phenotypes on the basis of metabolic health and underlying pathophysiology. Obesity and excess adiposity have a significant impact on fertility and reproductive health, with direct effects on the hypothalamic-pituitary-ovarian axis, the ovary and oocyte, and the endometrium. There are significant adverse pregnancy outcomes related to obesity, and excess weight gain before, during, and after pregnancy that can alter the lifelong risk for metabolically unhealthy obesity. Given the high prevalence and pervasive impact of obesity on reproductive health, there is a need for better and individualized care for reproductive-aged females that considers obesity phenotype, underlying pathophysiology, and effective and sustainable interventions to treat obesity and manage weight gain before, during, and after pregnancy.

Cellular heterogeneity and dynamics of the human uterus in healthy premenopausal women.

The human uterus is a complex and dynamic organ whose lining grows, remodels, and regenerates in every menstrual cycle or upon tissue damage. Here we applied single-cell RNA sequencing to profile more the 50,000 uterine cells from both the endometrium and myometrium of 5 healthy premenopausal individuals, and jointly analyzed the data with a previously published dataset from 15 subjects. The resulting normal uterus cell atlas contains more than 167K cells representing the lymphatic endothelium, blood endothelium, stromal, ciliated epithelium, unciliated epithelium, and immune cell populations. Focused analyses within each major cell type and comparisons with subtype labels from prior studies allowed us to document supporting evidence, resolve naming conflicts, and to propose a consensus annotation system of 39 subtypes. We release their gene expression centroids, differentially expressed genes, and mRNA patterns of literature-based markers as a shared community resource. We find many subtypes show dynamic changes over different phases of the cycle and identify multiple potential progenitor cells: compartment-wide progenitors for each major cell type, transitional cells that are upstream of other subtypes, and potential cross-lineage multipotent stromal progenitors that may be capable of replenishing the epithelial, stromal, and endothelial compartments. When compared to the healthy premenopausal samples, a postpartum and a postmenopausal uterus sample revealed substantially altered tissue composition, involving the rise or fall of stromal, endothelial, and immune cells. The cell taxonomy and molecular markers we report here are expected to inform studies of both basic biology of uterine function and its disorders. SIGNIFICANCE: We present single-cell RNA sequencing data from seven individuals (five healthy pre-menopausal women, one post-menopausal woman, and one postpartum) and perform an integrated analysis of this data alongside 15 previously published scRNA-seq datasets. We identified 39 distinct cell subtypes across four major cell types in the uterus. By using RNA velocity analysis and centroid-centroid comparisons we identify multiple computationally predicted progenitor populations for each of the major cell compartments, as well as potential cross-compartment, multi-potent progenitors. While the function and interactions of these cell populations remain to be validated through future experiments, the markers and their "dual characteristics" that we describe will serve as a rich resource to the scientific community. Importantly, we address a significant challenge in the field: reconciling multiple uterine cell taxonomies being proposed. To achieve this, we focused on integrating historical and contemporary knowledge across multiple studies. By providing detailed evidence used for cell classification we lay the groundwork for establishing a stable, consensus cell atlas of the human uterus.

A retrospective cross-sectional study: fresh cycle endometrial thickness is a sensitive predictor of inadequate endometrial thickness in frozen embryo transfer cycles.

BACKGROUND: The purpose of this study is to assess predictors of inadequate endometrial cavity thickness (ECT), defined as < 8 mm, in frozen embryo transfer (FET) cycles. METHODS: This is a retrospective cross-sectional study at an academic fertility center including 274 women who underwent their first endometrial preparation with estradiol for autologous FET in our center from 2001-2009. Multivariable logistic regression was performed to determine predictors of inadequate endometrial development in FET cycles. RESULTS: Neither age nor duration of estrogen supplementation were associated with FET endometrial thickness. Lower body mass index, nulliparity, previous operative hysteroscopy and thinner fresh cycle endometrial lining were associated with inadequate endometrial thickness in FET cycles. A maximum thickness of 11.5 mm in a fresh cycle was 80% sensitive and 70% specific for inadequate frozen cycle thickness. CONCLUSIONS: Previous fresh cycle endometrial cavity thickness is associated with subsequent FET cycle endometrial cavity thickness. Women with a fresh cycle thickness of 11.5 mm or less may require additional intervention to achieve adequate endometrial thickness in preparation for a frozen cycle.

Obesity and In Vitro Fertilization.

Obesity is a highly prevalent chronic disease with a significant effect on reproductive-age women. The clinical implications of obesity on fertility and pregnancy are well studied citing ovulatory dysfunction, hormonal imbalances, higher miscarriage rates, and increased maternal and neonatal risks. For this reason, many patients with obesity seek reproductive specialists to help build their families. Despite this literature base, the effect of weight loss interventions prior to assisted reproductive technology (ART) is lacking. This review aims to outline the impact of obesity on ART, specifically in vitro fertilization (IVF). Response differences to treatment protocols compared with normal weight counterparts, limitations of access to care, and the mixed results of weight-reduction strategies prior to fertility treatment will be addressed. The known data surrounding benefits of lifestyle modification, pharmacologic therapies, and surgical interventions for obesity prior to IVF are outlined and found to emphasize a need for further research to determine the optimal approach for infertility patients with obesity.

Proteomic analysis of human sperm reveals changes in protamine 1 phosphorylation in men with infertility.

OBJECTIVE: To perform a comprehensive assessment of protamine (P) isoforms and modifications in human sperm with the aim of identifying how P modifications and isoforms are altered in men with reduced sperm motility and low sperm count. DESIGN: Cross-sectional. SETTING: Academic medical center. PATIENTS: A total of 18 men with prior reported pregnancy and normozoospermia (normal sperm), 14 men from couples with infertility and asthenozoospermia (reduced sperm motility), and 24 men from couples with infertility and oligoasthenoteratozoospermia (low sperm count and motility and abnormal sperm morphology). INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Proteomic assessment using both top-down and bottom-up liquid chromatography mass spectrometry (MS) analysis. RESULTS: A total of 13 posttranslational modifications were identified on P1 and P2 using bottom-up MS, including both phosphorylation and methylation. Top-down MS revealed an unmodified and phosphorylated isoform of P1 and the 3 major isoforms of P2, HP2, HP3, and HP4. Protamine 1 phosphorylation was overall higher in men with male factor infertility compared with those with normal semen analysis (40.5% vs. 32.6). There was no difference in P posttranslational modifications or isoforms of P2 in men with normal vs. abnormal fertility. CONCLUSION: Human protamines bear a number of posttranslational modifications, with alterations in P1 phosphorylation noted in the setting of male factor infertility.

Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse protamine 1.

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by electrostatic interactions between DNA and the arginine-rich core of protamines. Phylogenetic analysis reveals several non-arginine residues conserved within, but not across species. The significance of these residues and their post-translational modifications are poorly understood. Here, we investigated the role of K49, a rodent-specific lysine residue in protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In sperm, alanine substitution (P1(K49A)) decreases sperm motility and male fertility-defects that are not rescued by arginine substitution (P1(K49R)). In zygotes, P1(K49A) leads to premature male pronuclear decompaction, altered DNA replication, and embryonic arrest. In vitro, P1(K49A) decreases protamine-DNA binding and alters DNA compaction and decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential for reproductive fitness.

The effects of type 1 diabetes on the hypothalamic, pituitary and testes axis.

Type 1 diabetes is an autoimmune disorder characterized by a lack of insulin production by the beta cells of the pancreas. This lack of insulin causes a variety of systemic effects on whole-body metabolism. Poorly managed type 1 diabetes can lead to cardiovascular disease, diabetic neuropathy, and diabetic retinopathy. Increasingly, even well-managed type 1 diabetic patients show damage to peripheral organs related to complications from the disease. The central role of insulin in energy homeostasis also renders it an important signaling factor in the reproductive tract. type 1 diabetes has now been demonstrated to cause defects in sperm and testes. The aim of this review is to present the known effects of insulin's role in the function of the male reproductive tract. These effects might be mediated through hormonal alterations in the hypothalamic pituitary gonadal axis or through the direct interaction of insulin on the testes and sperm cells. Although fertility complications also occur in type 2 diabetic males, this review will focus on the defects specifically linked with the lack of insulin seen in type 1 diabetes.

Discussion: 'Abnormal anal cytology in HIV-infected women' by Baranoski et al.

In the roundtable that follows, clinicians discuss a study published in this issue of the Journal in light of its methodology, relevance to practice, and implications for future research. Article discussed: Baranoski AS, Tandon R, Weinberg J, et al. Risk factors for abnormal anal cytology over time in HIV-infected women. Am J Obstet Gynecol 2012;207:107.e1-8.

Total fertilization failure with in vitro fertilization-intracytoplasmic sperm injection related to WEE2 mutation highlights emerging importance of genetic causes of in vitro fertilization failure.

Objective: To report a unique case of total fertilization failure (TFF) after in vitro fertilization with intracytoplasmic sperm injection related to homozygous WEE2 gene mutation and summarize the current literature and management of TFF. Design: Case report. Setting: Academic fertility center. Patients: A 25-year-old woman and her 35-year-old partner with a history of near-complete fertilization failure after 2 cycles of in vitro fertilization/intracytoplasmic sperm injection. Interventions: Consultation with medical and commercial genetic testing for WEE2, PLCZ1, and TLE6. Main Outcome Measures: Oocyte fertilization. Results: The patient was homozygous for WEE2 pathogenic variant impacting oocyte activation and resulting in infertility. Conclusions: In the setting of TFF, early consideration should be given to genetic testing to assist couples in clinical decision-making and help limit the financial and emotional burden associated with unsuccessful fertility intervention.

Obesity-induced follicular phase endometrial proteome dysregulation in a well-phenotyped population.

OBJECTIVE: Despite obesity's significant impact on reproduction, its influence on the physiology of the human endometrium is largely understudied. We hypothesized that endometrial proteomic differences exist between obese (OW; body mass index [BMI] ≥30 kg/m2) and normal-weight women (NWW; BMI, 18.5-24.9 kg/m2). DESIGN: Clinical cross-sectional study. SETTING: Academic Medical Center. PATIENT(S): Healthy, normally-cycling, 18 to 40-year-old women (n = 6 OW and n = 6 NWW). MAIN OUTCOME MEASURE(S): Participants underwent screening and midfollicular phase visits. Demographic and anthropometric characteristics, blood samples, ultrasounds, and follicular phase endometrial biopsies were collected. Proteomic analyses of endometrial samples (liquid chromatography-mass spectrometry) were performed. Proteins with ≥2-fold difference and a false discovery rate of <0.1 were considered statistically significant (Benjamini-Hochberg adjustment). RESULT(S): Reproductive hormone levels did not differ between the two groups. Mean BMI, serum leptin concentration, and bioelectrical impedance analysis indices of adiposity were higher in OW than in NWW. Histological examination of the endometrial samples confirmed normal-appearing endometrium in both OW and NWW. A total of 2,930 proteins were detected across all samples, with an average number of proteins per sample of 2,059 ± 482 in NWW and 2,437 ± 187 in OW. A total of 17 proteins were differentially expressed in OW vs. NWW; 2 were more abundant, whereas 15 were underexpressed in OW, including the progesterone receptor. CONCLUSION(S): In this well-phenotyped population of healthy women, obesity was associated with significant endometrial proliferative phase proteomic differences affecting the hormonal and immunologic pathways. These could contribute to an increased risk of menstrual bleeding abnormalities and create an altered environment for future luteinization.

Obesity-related alterations in protein expression in human follicular fluid from women undergoing in vitro fertilization.

OBJECTIVE: To compare the proteomic composition of follicular fluid from women with normal weight vs. women with obesity but without a history of polycystic ovary syndrome or known ovarian dysfunction undergoing in vitro fertilization. DESIGN: Cross-sectional. SETTING: Academic medical center. PATIENT(S): Eight women with normal weight and 8 women with obesity undergoing in vitro fertilization and without a history of polycystic ovary syndrome, ovulatory dysfunction, diminished ovarian reserve, or known endometriosis were included in the analysis. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): Proteomic assessment using liquid chromatography-mass spectrometry analysis. RESULT(S): The mean age of women with normal weight was similar to that of women with obesity (32.9 vs. 32.6 years, not significant). The mean body mass index of women with normal weight was 21.2 kg/m2 compared with a body mass index of 37.1 kg/m2 in women with obesity. A total of 1,174 proteins were identified with ≥2 peptides present. Twenty-five proteins were found to be significantly altered in the follicular fluid from women with obesity. Of these 25 proteins, 19 were up-regulated and 6 were down-regulated. Notably, C-reactive protein was 11-fold higher in the follicular fluid from women with obesity than in the follicular fluid from women with normal weight. CONCLUSION(S): Obesity is associated with dysregulation at the level of the follicle, including alterations in proteins related to inflammation and metabolism. These include proteins with emerging roles in energy homeostasis and follicular regulation.

Outcomes of subchorionic hematoma-affected pregnancies in the infertile population.

OBJECTIVE: To determine the implications of an incidentally noted subchorionic hematoma on pregnancy outcomes in the infertile population. METHODS: Retrospective cohort study at a tertiary care, university-based facility. All patients with intrauterine pregnancy on initial obstetric ultrasound presenting to an infertility clinic between January 2015 and March 2018 (n = 1210), regardless of treatment cycle, were included. Nonviable pregnancies were excluded. The main outcome measured was association between subchorionic hematoma and first trimester miscarriage. RESULTS: The prevalence of subchorionic hematoma was 12.5% (n = 151) and did not differ by type of fertility treatment. There was no association between subchorionic hematoma and first trimester miscarriage; however, among patients with subchorionic hematoma, those who reported both bleeding and cramping had an increased probability of miscarriage compared to those without symptoms (0.62 vs. 0.12, P <0.001). The live birth rate in this sample was 81.3% and there were no statistically significant differences in pregnancy outcomes between those with and without subchorionic hematoma. CONCLUSION: Among an infertile population, there was no increased risk of miscarriage when subchorionic hematoma was seen on early ultrasound; however, when patients noted both vaginal bleeding and cramping, their probability of miscarriage was significantly increased.

Sperm centriole assessment identifies male factor infertility in couples with unexplained infertility - a pilot study.

Unexplained infertility affects about one-third of infertile couples and is defined as the failure to identify the cause of infertility despite extensive evaluation of the male and female partners. Therefore, there is a need for a multiparametric approach to study sperm function. Recently, we developed a Fluorescence-Based Ratiometric Analysis of Sperm Centrioles (FRAC) assay to determine sperm centriole quality. Here, we perform a pilot study of sperm from 10 fertile men and 10 men in couples with unexplained infertility, using three centriolar biomarkers measured at three sperm locations from two sperm fractions, representing high and low sperm quality. We found that FRAC can identify men from couples with unexplained infertility as the likely source of infertility. Higher quality fractions from 10 fertile individuals were the reference population. All 180 studied FRAC values in the 10 fertile individuals fell within the reference population range. Eleven of the 180 studied FRAC values in the 10 infertile patients were outliers beyond the 95% confidence intervals (P = 0.0008). Three men with unexplained infertility had outlier FRAC values in their higher quality sperm fraction, while four had outlier FRAC values in their lower quality sperm fraction (3/10 and 4/10, P = 0.060 and P = 0.025, respectively), suggesting that these four individuals are infertile due, in part, to centriolar defects. We propose that a larger scale study should be performed to determine the ability of FRAC to identify male factor infertility and its potential contribution to sperm multiparametric analysis.