Microarray evidence that 8-cell human embryos express some hormone family members including oxytocin.

OBJECTIVE: This study is to discover hormone pathways active in early cleaving human embryos. METHODS: A list of 152 hormones and receptors were compiled to query the microarray database of mRNAs in 8-cell human embryos, two lines of human embryonic stem cells plus human fibroblasts before and after induced pluripotency. RESULTS: Over half of the 152 hormones and receptors were silent on the arrays of all cell types, and more were detected at high or moderate levels on the 8-cell arrays than on the pluripotent cell or fibroblast arrays. Eight hormone family genes were uniquely detected at least 22-fold higher on the 8-cell arrays than the stem cell arrays: AVPI1, CCK, CORT, FSTL4, GIP, GPHA2, OXT, and PPY suggesting novel roles for these proteins in early development. Oxytocin was detected by pilot immunoassay in culture media collected from Day 3 embryos. Robust detection of CRHR1 and EPOR suggests the 8-cell embryo may be responsive to maternal CRH and EPO. The over-expression of POMC and GHITM suggests POMP peptide products may have undiscovered roles in early development and GHITM may contribute to mitochondrial remodeling. Under-detected on the 8-cell arrays at least tenfold were two key enzymes in steroid biosynthesis, DHCR24 and FDPS. CONCLUSIONS: The 8-cell human embryo may be secreting oxytocin, which could stimulate its own progress down the fallopian tube as well as play a role in early neural precursor development. The 8-cell embryo does not synthesize reproductive steroid hormones. As previously reported for growth factor families, the early embryo over-expresses more hormones than hormone receptors.

A new approach to postvasectomy semen analyses eliminates the need to evaluate a fresh specimen.

BACKGROUND: According to current guidelines, confirmation that vasectomy results in sterility depends on microscopic examination of postvasectomy semen for the presence of spermatozoa. Guidelines established in 2012 require examination of a fresh specimen within 2 h of collection, which necessitates the patient making an appointment with either the surgeon's office or a licensed clinical laboratory. Twenty-five to 42% of patients fail to comply with postvasectomy semen analysis (PVSA). OBJECTIVES: To determine if an at-home semen collection kit can substitute for the evaluation of a fresh specimen and improve patient compliance with postvasectomy spermatozoa assessment. MATERIALS AND METHODS: The kit contains a patented aldehyde-fixative that maintains spermatozoa and semen cells in suspension for quantitation. Patients order a PVSA kit to be delivered to their home and can collect a semen specimen and return it to the laboratory through regular US mail. RESULTS: From January 2011 through December 2018, 6096 men undergoing vasectomy by 184 urologists in 33 states in the US ordered PVSA kits, of which 5408 (89%) returned at least one for analysis. Of those, 398 men (7.4%) returned the first kit with greater than 10,000 spermatozoa/ml within a year of vasectomy, of which only 4.4% contained greater than 100,000 spermatozoa/ml 12 weeks postsurgery. This suggests that fewer than 5% of postvasectomy patients might need follow-up fresh semen analyses, greatly easing the logistical burden of PVSA. Ninety percent of surgeons returning a patient satisfaction questionnaire said their patients "never" complained about using PVSA kits. DISCUSSION AND CONCLUSION: These data support the adoption of a new standard for PVSA that does not involve an initial evaluation of a fresh semen specimen.

Retroviruses and reproduction revisited.

Thanks to effective anti-HIV medications, deaths from acquired immunodeficiency disease (AIDS) have plummeted, although the incidence of new HIV infections has decreased little, approximately 36,000 annually in the USA. The CDC estimates 1.1 million persons, mostly men, are living with HIV in the USA, with approximately 14% unaware they are infected. Since the global blood supply is essentially free of HIV today, infected semen is fueling the pandemic (88% of new infections in the USA), with needle sharing among IV drug abusers (7% of new US infections) and female to male transmission (5% of new infections) accounting for the balance. In spite of the importance to disease prevention and strategies for safe conception, semen transmission of HIV is not well understood. Because anti-HIV therapy does not eliminate HIV from semen, the Centers for Disease Control (CDC) for the past 25 years has espoused condom use as the safest approach to prevent HIV transmission, as well as other sexually transmitted diseases. A few months ago, however, an MMWR was circulated by the CDC that suggested condomless sex might be safe if the HIV-infected partner's medications achieved an undetectable viral load in his blood. This new opinion was based on reports by three teams of investigators cited in the MMWR: "All three studies observed no HIV transmission to the uninfected partner while the partner with HIV was virologically suppressed with ART." Unfortunately, this CDC statement does not fully describe the data presented in the studies, and abandoning condom use puts uninfected partners, including women seeking to conceive, at risk for infection by HIV and other STDs.

Early human embryos are naturally aneuploid-can that be corrected?

Aneuploidy is common and may be a natural occurrence in early human embryos. Selecting against embryos containing aneuploid cells for embryo transfer has been reported to increase clinical pregnancies per transfer in some studies, but not others. Some aneuploidy is due to misallocation of chromosomes during meiosis, in either the egg or sperm, but most aneuploidy is due to misallocation of chromosomes during mitoses after fertilization. Big questions are as follows: Why does this happen? How much aneuploidy in a preimplantation embryo is compatible with normal fetal development? Is aneuploidy increased by in vitro culture, and/or could it be prevented or corrected in the IVF lab?

Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells.

Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that it is an independent circadian rhythm-competent equivalence group poised to signal its environment, defend against maternal immune rejection, and begin the rapid commitment events of early embryogenesis.

Cytomegalovirus and human immunodeficiency virus in semen of homosexual men.

OBJECTIVE: To assess the accuracy of serology to predict the presence of cytomegalovirus (CMV) in semen of homosexual men without and with HIV coinfection. DESIGN: Semen CMV was detected by electron microscopy and by polymerase chain reaction (PCR) amplification; paired serum was tested for CMV IgG/IgM. Semen HIV was detected by reverse transcription-PCR. SETTING: Licensed clinical and research laboratory. PATIENT(S): Sixty-eight men. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Frequency of CMV and HIV in semen. RESULT(S): Cytomegalovirus was detected by electron microscopy in 3 of 10 specimens examined. Forty-six (89%) of 52 HIV-infected men were seropositive for CMV by combined assay for IgG/IgM; two more (48 of 52, 92%) were seropositive for CMV IgG by separate assay; 25 (48%) of the HIV-infected men had PCR-detectable CMV DNA in at least one semen specimen, 22 of whom (42%) had CMV in all specimens. Nineteen (13%) of the 150 specimens tested positive for HIV, whereas 67 (45%) tested positive for CMV; seven specimens tested positive for both CMV and HIV. Cytomegalovirus, but not HIV, detection in semen correlated with decreased CD4(+) lymphocytes in peripheral blood (<700/µL) but was not accurately predicted by serology, leukocytospermia, or age. CONCLUSION(S): Cytomegalovirus in semen is not accurately predicted by serology. Sperm banking needs to include direct assessment of CMV in semen specimens. Strategies to eliminate CMV from semen specimens are needed to alleviate the risk of virus transmission.

Timing is everything in the human embryo.

A noninvasive imaging method for predicting how human embryos will develop may improve the success and safety of in vitro fertilization.

Genome-wide microarray evidence that 8-cell human blastomeres over-express cell cycle drivers and under-express checkpoints.

PURPOSE: To understand cell cycle controls in the 8-Cell human blastomere. METHODS: Data from whole human genome (43,377 elements) microarray analyses of RNAs from normal 8-Cell human embryos were compiled with published microarrays of RNAs from human fibroblasts, before and after induced pluripotency, and embryonic stem cells. A sub database of 3,803 genes identified by high throughput RNA knock-down studies, plus genes that oscillate in human cells, was analyzed. RESULTS: Thirty-five genes over-detected at least 7-fold specifically on the 8-Cell arrays were enriched for cell cycle drivers and for proteins that stabilize chromosome cohesion and spindle attachment and limit DNA and centrosome replication to once per cycle. CONCLUSIONS: These results indicate that 8-cell human blastomere cleavage is guided by cyclic over-expression of key proteins, rather than canonical checkpoints, leading to rapidly increasing gene copy number and a susceptibility to chromosome and cytokinesis mishaps, well-noted characteristics of preimplantation human embryos.

Effect of PRL on in vitro follicle growth, in vitro oocyte maturation, fertilization and early embryonic development in mice.

Prolactin (PRL), along with other hormones, plays a role in oocyte maturation, fertilization, and early embryonic development in mammals. In order to investigate the role of PRL on in vitro oocyte maturation from early follicular growth stages, as well as on fertilization and early embryonic development, we cultured preantral mouse follicles with and without PRL, followed by fertilization of the in vitro matured oocytes. Prolactin significantly improved the rate of oocyte maturation, fertilization, and early embryo development. Four isoforms of PRL-Receptor (R) have been found in whole ovaries of mice: one long (PRL-RL) and three short (-RS(1), -RS(2), and -RS(3)). We examined expression of the four PRL-R isoforms in preantral follicles, in cumulus-oocyte complexes (COCs) and in germinal vesicle GV stage oocytes by RT-PCR. Prolactin-RL, -RS(2) and -RS(3) mRNA, but not -RS(1), were expressed in preantral follicles, COCs, and GV stage oocytes. Our results indicate the prolactin pathway is functional in early preantral follicles, in COCs and in GV stage oocytes, and promotes oocyte maturation, meiosis, fertilization, and early embryonic development.

Evidence that human blastomere cleavage is under unique cell cycle control.

PURPOSE: To understand the molecular pathways that control early human embryo development. METHODS: Improved methods of linear amplification of mRNAs and whole human genome microarray analyses were utilized to characterize gene expression in normal appearing 8-Cell human embryos, in comparison with published microarrays of human fibroblasts and pluripotent stem cells. RESULTS: Many genes involved in circadian rhythm and cell division were over-expressed in the 8-Cells. The cell cycle checkpoints, RB and WEE1, were silent on the 8-Cell arrays, whereas the recently described tumor suppressor, UHRF2, was up-regulated >10-fold, and the proto-oncogene, MYC, and the core element of circadian rhythm, CLOCK, were elevated up to >50-fold on the 8-Cell arrays. CONCLUSIONS: The canonical G1 and G2 cell cycle checkpoints are not active in totipotent human blastomeres, perhaps replaced by UHRF2, MYC, and intracellular circadian pathways, which may play important roles in early human development.

Detection and identification of bacterial DNA in semen.

OBJECTIVE: To detect and identify bacteria in semen by sequencing polymerase chain reaction (PCR)-amplified ribosomal RNA gene regions (rDNAs). DESIGN: Bacterial rDNAs were detected by PCR amplification of semen DNA. Conditions were adjusted to detect only abundant organisms, no fewer than 20,000 bacteria/mL of semen. SETTING: Clinical andrology laboratory and academic research laboratories. PATIENT(S): Men undergoing fertility evaluation (n = 29) or vasectomy (n = 5). INTERVENTION(S): None. MAIN OUTCOME MEAURE(S): Frequency of bacterial rDNA-positive specimens, relationship of rDNAs to bacteria in GenBank, and correlation with semen cells. RESULT(S): Twenty-five (56%) of the specimens from 22 (65%) of the men were positive. A total of 141 bacterial rDNA sequences were compared with GenBank data for identification. The largest group matched gram-positive anaerobic cocci (Peptoniphilis, Anaerococcus, Finegoldia, Peptostreptococcus spp.) in 13 specimens, followed by Corynebacterium spp. in 10 specimens, Staphylococcus, Lactobacillus, and Streptococcus spp. in 7 specimens each, Pseudomonas spp. in 4 specimens, and Haemophilus and Acinetobacter spp. in 2 specimens each. The rDNA-positive specimens averaged 59 +/- 13 million sperm/mL, 46 +/- 5% of which were motile, not statistically different from the rDNA-negative specimens (77 +/- 16 million/mL, 47 +/- 5% motile). Normal sperm forms were lower in the rDNA-positive (10 +/- 1.1%) than in the rDNA-negative specimens (22 +/- 2%), and lymphocytes/monocytes were fivefold lower in the rDNA-positive specimens (0.4 +/- 0.2 million/mL) than in the negative specimens (1.9 +/- 0.7 million/mL). CONCLUSION(S): Abundant bacteria in semen are not commensal, arise from infection in the male genitourinary tract, may influence fertility, and may reflect an inadequate cellular immune response.

Isolation of human immunodeficiency virus type 1 from semen and vaginal fluids.

Semen and vaginal fluids transmit HIV infection. The virus is present as cell-free particles and as infected cells. Isolation of infectious virus from both genital tract fluids poses unique problems. Vaginal fluids are heavily contaminated with normal bacterial flora, and seminal plasma is cytotoxic to peripheral blood mononuclear cells. Adaptations of routine laboratory procedures have been developed to largely overcome these problems, allowing the culture and characterization of genital-tract HIV.

Detection of drug-resistant HIV-1 strains.

Human immunodeficiency virus (HIV-1) encodes proteins essential to its replication cycle. Reverse transcriptase, protease, and viral envelope gp120 are three proteins that have been targeted for antiviral drug development. Eleven inhibitors of reverse transcriptase, seven inhibitors of protease, and one inhibitor of viral envelope binding have been approved for use. Antiretroviral therapy has reversed the mortality rate of HIV-infected persons, but over time, therapy-resistant virus variants may outgrow. A large body of information is now available to relate specific amino acid sequences in the resistant variants to specific drug regimens. Designing therapy to compensate for virus resistance results in improved patient outcomes. The advent of microsequencing technologies paved the way for direct sequencing of DNA products generated by polymerase chain reactions, thus dramatically lowering the cost of HIV gene sequencing. Designing therapy according to genetic analysis of HIV variants will not only also improve clinical outcome, but will also deter the transmission of drug-resistant strains.

Tissue-specific populations of leukocytes in semen-producing organs of the normal, hemicastrated, and vasectomized mouse.

Semen HIV is separate and distinct from blood HIV and work has revealed that seminal plasma HIV particles do not arise from infected cells in semen. These findings indicate that semen-producing organs contain multiple, separate populations of HIV host cells. To test this hypothesis, we have examined leukocytes in semen-producing organs of male mice. Cells expressing F4/80 (tissue-specific macrophage marker) were abundant in testicular interstitium and as dendritic-like cells in the lumenal epithelium of the epididymis, especially the initial segment. Cells expressing CD45 (panleukocyte marker) were found rarely in the testicular interstitium, commonly in epididymal epithelium, were most abundant in the interstitium of the epididymis, and were more readily released from minced tissues than were F4/80(+) cells. Unlike the testis and epididymis, F4/80(+) cells in seminal vesicles also appeared to be CD45(+). Seminal vesicle leukocytes were restricted to the epithelium surrounding the lumen and were not released by mincing. CD11b (monocyte/B cell marker) was detected in testicular and seminal vesicle interstitium, but not in the epididymis. Hemicastration and vasectomy caused a limited redistribution of the leukocytes. These findings confirm the existence of tissue-specific populations of leukocytes in semen-producing organs and indicate that some populations are highly tissue adherent. The regionalized, tissue-adherent macrophages in the testicular interstitium, the initial segment of the caput epididymis, and the seminal vesicle epithelium suggest the existence of reservoirs of HIV-infected cells in humans that could contribute virus particles, but not infected cells, to semen and possibly blood.

Seminal plasma induces programmed cell death in cultured peripheral blood mononuclear cells.

Immunosuppressive properties of seminal plasma inhibit the recovery of infectious HIV from semen, and led to the view early in the pandemic that semen HIV was transmitted principally by infected semen cells. More recent studies have revealed significant titers of HIV RNA in seminal plasma, however, even from men receiving successful antiviral therapy. Thus, studies of infectious HIV in seminal plasma are important to understanding sexual transmission and response to therapy. The present studies were undertaken to determine whether seminal plasma immunosuppression is mediated by the induction of programmed cell death (PCD). Peripheral blood mononuclear cells (PBMCs) were cultured without or with phytohemagglutinin and seminal plasma from normal donors, or men postvasectomy, or seminal vesicle protein collected at surgery. PBMC survival was measured at 3, 6, and 18 hr of culture; cells were examined for evidence of PCD by uptake of the fluorescent dye YO-PRO, and for fragmented nuclear DNA by the TUNEL assay. Approximately 90% of PBMCs cultured with seminal plasma from intact or vasectomized men were lost during 18 hr of culture; seminal vesicle protein did not induce cell loss. PCD assays were positive for PBMCs exposed to the seminal plasma, and negative for PBMCs cultured with seminal vesicle protein. Serum was not required for PCD induction. A 3-hr pulse with seminal plasma was sufficient to initiate PCD. These findings indicate that PCD induction accounts for the cytotoxic properties of semen, that the PCD is not the result of semen amine oxidases, and either that substances produced by seminal vesicles only at ejaculation, or by the prostate, are responsible for PCD induction.

Overseeing research on therapeutic cloning: a private ethics board responds to its critics.

Advanced Cell Technology's Ethics Advisory Board has been called window dressing for a corporate marketing plan. But the scientists and managers have paid attention, and the lawyers have gone along.