Mixture Of Sperm Research Articles

Distribution, Population Systems, and Peculiarities of Hybrid Gametogenesis in Water Frogs (<i>Pelophylax esculentus</i> complex) in the Dniester River Valley (Moldova)

Hybridogenesis is well-known attributes of water frogs of the genus Pelophylax. The P. esculentus complex consists of two parental species, P. lessonae and P. ridibundus, as well as their hemiclonal (sometimes meroclonal) hybrid, P. esculentus. DNA flow cytometry, genetic and morphometric analyses were used to examine water frog compositions in 27 localities (n = 212) throughout the Dniester River valley in Moldova. Two species were revealed in the studied region (42% individuals were P. esculentus and 58% P. ridibundus). All frogs proved to be diploid. We registered the first species in 74% localities and the second in 78%. Populations, where we found only P. ridibundus occurred in 26% localities and only P. esculentus in 22%, while mixed population systems in 52%. Both species were usually observed in open water bodies and rivers that flow down through agricultural land and urban areas. In populations of P. esculentus the number of males strongly exceeded (90%) the number of females. The half of studied hybrid males was sterile, and most of fertile hybrid males produced sperm with the genome of P. lessonae. Several hybrid males gave the mixture of sperm with genomes of P. lessonae and P. ridibundus (amphispermy) and only one male with the P. ridibundus genome. Both species had the mitochondrial DNA of P. ridibundus. Based on nuclear markers, in P. esculentus and P. ridibundus we revealed the presence of alleles of the closely related Anatolian species P. cf. bedriagae. This might affect the successful reproduction of hybridogenetic P. esculentus. However, comparison of percentage of sterile males and alleles of P. cf. bedriagae among hybrids showed no correlation.

Application of piezoactuators in the technology of low-temperature preservation of fish reproductive cells

The sperm of Russian sturgeon (Acipenser gueldenstaedtii Brandt et Ratzeburg, 1833) was the research object. Acoustic-mechanical influence on sturgeon semen improves fish-biological and reproductive indices of the seminal fluid. The most optimal indices for reproductive cells of male Russian sturgeon are 500 Hz, 90 V with the exposure duration of 1 minute. It has been shown that when a piezo-actuator with a frequency of 500 Hz and an amplitude of 90 V is applied to the sperm mixture with the cryoprotectant, ice crystals are formed later, which may influence the survival of cells after defrosting.

DNA profiling of single sperm cells after whole genome amplification

Intimate swabs taken for examination in sexual assault cases typically yield mixtures of sperm and epithelial cell types. While powerful, differential extraction protocols to overcome such cell type mixtures by separate lysis of epithelial cells and spermatozoa can still prove ineffective, in particular if only few sperm cells are present or if swabs contain sperm from more than one individual leading to complex low level DNA mixtures. A means to avoid such mixtures consists in the analysis of single micromanipulated sperm cells. However, the quantity of DNA from single sperm cells is not sufficient for conventional STR analysis. Here, we describe a simple method for micromanipulating individual sperm cells from intimate swabs and show that whole genome amplification can generate sufficient amounts of DNA from single cells for subsequent DNA profiling. We recovered over 80% of alleles of haploid autosomal STR profiles from the majority of individual sperm cells. Furthermore, we demonstrate that in mixtures of sperm from two contributors, Y-STR and X-STR profiles of individual sperm cells can be used to sort the haploid autosomal profiles to develop the diploid consensus STR profiles of the individual donors. Finally, by analyzing single sperm cells from mock sexual assault swabs with one or two sperm donors, we showed that our protocols enabled the identification of the unknown male contributors.

Sexual maturity, lack of partner choice and sperm precedence in the promiscuous ladybird beetle Eriopis connexa (Germar): Who is my father?

Ladybird beetles present as key predator of many agricultural pests. Among them, Eriopis connexa stands out due some important traits for pest management programs such as common occurrence, population selected for resistance to pyrethroid insecticides, and possibility to be commercially available by rearing using alternative prey. Despite that, little is known about the role of polyandry and its effect on paternity in this species. Ladybird beetles engage in multiple matings, raising questions about the benefits of polyandry and paternity. We studied the selection of mating pair, sperm precedence, and offspring paternity by performing experiments on (1) the age of sexual maturity, (2) the frequency and refractory mating behavior within a photophase period, (3) the preference of insecticide-susceptible females to mate with either susceptible or resistant male phenotypes; and (4) the effect of multiple matings, on progeny paternity. Sexual maturity in E. connexa was found to begin within 3 days of adult emergence for both sexes, but fully developed after 5 days. The highest frequency of mating exhibited by sexual mature pairs occurred within first hour of pairing and the phenotype for insecticide resistance did not affect the choice of a mating partner. The resistance trait marker in the resistant beetle phenotype indicates that progeny paternity results from a mixture of available sperm and do not depend on mating order. These data suggest that released beetles would have a simple prospect for progeny production for both insecticide resistant and susceptible beetles.

Analysis of Additional Methods of purification of Mixtures while Conducting Differential Lysis under Sexual Crimes

The article is focused on the problem of studying mixed biological traces during the research of the growing number and severity of sexual crimes. The presence of blood and epithelial cells with a small amount of sperm on things removed at the crime scene and smears from the victim’s body, complicates the molecular and genetic examination and the likelihood of obtaining a quality DNA profile from the sperm of the rapist. Therefore, the author has defined chemical basis of the standard method of differential lysis and the correlation of the components of the mixture of epithelial cells and sperm, which can help to get mixed and pure DNA profiles while amplify using the sets AmpFlSTR® Identifiler® PCR Amplification Kit, GlobalFiler STR PCR Amp Afication ™ PCR Amplification Kit. The author has analyzed literature data on the developed and perspective methods and additional stages of the differential lysis technique for purification of the mixture and aimed at sorting and reducing the number of epithelial cells. Chemical methods of mixture purification have been studied, including additional washing and incubation of the mortar, use of DNases, SDS lysis buffer and magnetic sorting with the use of antibodies (MOSPD3 and RH-20), and physical methods such as laser microdissection, acoustic lysis, hydrodynamic effect in small Reynolds numbers, microchips with additional integration of Cialyl Lewis oligosaccharide sequence and DEPArray technology. The main advantages and disadvantages of the procedures, the possibility of combining with other types, the impact on subsequent stages of molecular and genetic examination and the possibility of automating the research process have been provided for the methods. For the methods, the author has studied the probability of contamination during the procedure and the possibility of using for obsolete traces of sperm, where the destruction of sperm structure occurred. The author has indicated the possibility of automation of physical systems for forensic examination of molecular and genetic analysis by integrating the stages of sample preparation, cell counting and DNA isolation on a single device, which will reduce the time of the analysis of research objects in the future. The analysis of the literature data showed an improvement in the quality of DNA profiles while using the above methods, so it has been offered to use the presented methods for testing in expert practice.

Longer and faster sperm exhibit better fertilization success in Japanese quail

In birds, sperm storage tubules (SST) located in the utero-vaginal junction are thought to be a site of sperm selection; however, the exact mechanism of sperm selection is poorly understood. Here, we investigated sperm entry into the SST and subsequent fertilization success under a competitive situation created by artificial insemination of a sperm mixture obtained from 2 males. We employed 2 quail strains, a wild-type and a dominant black (DB) type, as this allows easy assessment of paternity by feather coloration. We found paternity of embryos was biased toward DB males when a sperm mix with similar sperm numbers from the 2 males strains was artificially inseminated into females. Our novel sperm staining method with 2 different fluorescent dyes showed that the DB-biased fertilization was because of the better ability of DB sperm to enter the SST. Moreover, we found that DB sperm had a longer flagellum and midpiece. These characteristics probably allow sperm to swim faster in a high viscosity medium, which may be a similar environment to the lumen of the female reproductive tract. Our results indicated that sperm competition occurs to win a place in the SST and that filling the SST with their own spermatozoa is a critical step to achieve better fertilization success for the male Japanese quail.

Back Cover: Distinct and independent dielectrophoretic behavior of the head and tail of sperm and its potential for the safe sorting and isolation of rare spermatozoa

DOI: 10.1002/elps.201800437 The cover picture shows a heterogeneous mixture of live sperm, dead sperm (stained with red fluorescent dye) and other cell types enter the microfluidic device. A focusing electrode guides them to the di-electrophoresis-based sorting electrode pair. The unwanted cells and debris are repelled by the electrode and leave via the upper channel while the live sperm`s tails (both motile and immotile) are pulled onto the electrodes guiding them to an alternate exit port. The inset shows the electric field intensity distribution. The fields are highest in the gap between the electrodes where the tail is trapped while the head on the other hand is distanced from the potentially harmful electric fields to enable safe sorting.

Protein-Level Interactions as Mediators of Sexual Conflict in Ants*

All social insects with obligate reproductive division of labor evolved from strictly monogamous ancestors, but multiple queen-mating (polyandry) arose de novo, in several evolutionarily derived lineages. Polyandrous ant queens are inseminated soon after hatching and store sperm mixtures for a potential reproductive life of decades. However, they cannot re-mate later in life and are thus expected to control the loss of viable sperm because their lifetime reproductive success is ultimately sperm limited. In the leaf-cutting ant Atta colombica,, the survival of newly inseminated sperm is known to be compromised by seminal fluid of rival males and to be protected by secretions of the queen sperm storage organ (spermatheca). Here we investigate the main protein-level interactions that appear to mediate sperm competition dynamics and sperm preservation. We conducted an artificial insemination experiment and DIGE-based proteomics to identify proteomic changes when seminal fluid is exposed to spermathecal fluid followed by a mass spectrometry analysis of both secretions that allowed us to identify the sex-specific origins of the proteins that had changed in abundance. We found that spermathecal fluid targets only seven (2%) of the identified seminal fluid proteins for degradation, including two proteolytic serine proteases, a SERPIN inhibitor, and a semen-liquefying acid phosphatase. In vitro, and in vivo, experiments provided further confirmation that these proteins are key molecules mediating sexual conflict over sperm competition and viability preservation during sperm storage. In vitro, exposure to spermathecal fluid reduced the capacity of seminal fluid to compromise survival of rival sperm in a matter of hours and biochemical inhibition of these seminal fluid proteins largely eliminated that adverse effect. Our findings indicate that A. colombica, queens are in control of sperm competition and sperm storage, a capacity that has not been documented in other animals but is predicted to have independently evolved in other polyandrous social insects.

Pressure-based alkaline lysis with immunocapture, a method for enhanced recovery in differential extraction.

A pressure-based protocol for differential extraction has been optimized to provide a rapid and selective alternative to conventional differential extraction techniques with the advantage of an increased recovery of genomic DNA. The protocol involves treating cotton swabs containing mixtures of sperm and vaginal epithelial cells with two sets of pressure treatment using a Barocycler® NEP 2320 in alkaline conditions. This first step quantitatively and selectively removes female epithelial cells. The cotton swab is removed and further treated with alkali at 95°C for the removal of sperm cell DNA. The resultant solution provides a clean male profile at a 20:1 cell ratio. Furthermore, the inclusion of a pretreatment step involving immunomagnetic cell capture of the female cells, permits nearly complete isolation of male sperm cells at cell ratios of up to 200:1 female to male cells.

Fluorescence- and magnetic-activated cell sorting strategies to separate spermatozoa involving plural contributors from biological mixtures for human identification.

No effective method has been developed to distinguish sperm cells originating from different men in multi-suspect sexual assault cases. Here we combined MACS and FACS to isolate single donor sperm cells from forensic mixture samples including female vaginal epithelial cells and sperm cells from multiple contributors. Sperms from vaginal swab were isolated by MACS using FITC-conjugated A kinase anchor protein 3 (AKAP3) antibody; target individual sperm cells involving two or three donors were separated by FACS using FITC-labeled blood group A/B antigen antibody. This procedure was further tested in two mock multi-suspect sexual assault samples and one practical casework sample. Our results showed that complete single donor STR profiles could be successfully obtained from sperm/epithelial cell and sperm mixtures from two contributors. For unbalanced sperm/epithelial cells and sperm cells mixtures, sensitivity results revealed that target cells could be detected at as low as 1:32 and 1:8 mixed ratios, respectively. Although highly relies on cell number and blood types or secretor status of the individuals, this procedure would still be useful tools for forensic DNA analysis of multi-suspect sexual assault cases by the combined use of FACS and MACS based on sperm-specific AKAP3 antigen and human blood type antigen.

Sperm competition risk drives plasticity in seminal fluid composition.

BackgroundEjaculates contain a diverse mixture of sperm and seminal fluid proteins, the combination of which is crucial to male reproductive success under competitive conditions. Males should therefore tailor the production of different ejaculate components according to their social environment, with particular sensitivity to cues of sperm competition risk (i.e. how likely it is that females will mate promiscuously). Here we test this hypothesis using an established vertebrate model system, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our study tests for the first time how both sperm and seminal fluid components of the ejaculate are tailored to the social environment.ResultsOur quantitative proteomics analysis reveals that the relative production of different proteins found in seminal fluid – i.e. seminal fluid proteome composition – differs significantly according to cues of sperm competition risk. Using a conservative analytical approach to identify differential expression of individual seminal fluid components, at least seven of 31 secreted seminal fluid proteins examined showed consistent differences in relative abundance under high versus low sperm competition conditions. Notably three important proteins with potential roles in sperm competition – SVS 6, SVS 5 and CEACAM 10 – were more abundant in the high competition treatment groups. Total investment in both sperm and seminal fluid production also increased with cues of heightened sperm competition risk in the social environment. By contrast, relative investment in different ejaculate components was unaffected by cues of mating opportunities.ConclusionsOur study reveals significant plasticity in different ejaculate components, with the production of both sperm and non-sperm fractions of the ejaculate strongly influenced by the social environment. Sperm competition risk is thus shown to be a key factor in male ejaculate production decisions, including driving plasticity in seminal fluid composition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0197-2) contains supplementary material, which is available to authorized users.

Separation Patterns of Semen Mixtures: The Major-Minor Impact

The differential extraction procedure first described by Gill et al. [1] refers to the process by which the DNA from epithelial cells and sperm cells can be separated into different fractions. Compared with other methods such as flow cytometry [2] and laser microdissection [3,4], differential extraction is relatively quick and low-cost. In the investigation of cases of sexual assault, semen stains on vaginal swabs and clothing or bedding items provide the most incriminating evidence. The elucidation of the perpetrator or contributor by DNA analysis is most often straightforward in a single-rape scenario. However, in a gang-rape situation the elucidation process becomes more complex due to the presence of multiple contributors in the semen stains. Semen stains on clothing or bedding items in a gang-rape could invariably consist entirely of sperm DNA from multiple perpetrators. When differential extraction is performed on these stains, the DNA differential profiles in both epithelial and sperm fractions would hence be of sperm origin. In this study, semen from different sources was separately dried on cloth and FTA cards. FTA cards are essentially cellulose paper with protective matrix to archive DNA [5]. The DNA on FTA cards would benchmark as non-degraded DNA. Differential extraction was carried out on imbalanced mixtures of sperm with an excess of DNA from one source, purportedly the major contributor. The differential profiles were analyzed to determine the separation patterns.

Separation of sperm and epithelial cells based on the hydrodynamic effect for forensic analysis.

In sexual assault cases, forensic samples are a mixture of sperm from the perpetrator and epithelial cells from the victim. To obtain an independent short tandem repeat (STR) profile of the perpetrator, sperm cells must be separated from the mixture of cells. However, the current method used in crime laboratories, namely, differential extraction, is a time-consuming and labor-intensive process. To achieve a rapid and automated sample pretreatment process, we fabricated a microdevice for hydrodynamic and size-based separation of sperm and epithelial cells. When cells in suspension were introduced into the device's microfluidic channels, they were forced to flow along different streamlines and into different outlets due to their different diameters. With the proposed microdevice, sperm can be separated within a short period of time (0.5 h for a 50-μl mock sample). The STR profiles of the products in the sperm outlet reservoir demonstrated that a highly purified male DNA fraction could be obtained (94.0% male fraction). This microdevice is of low-cost and can be easily integrated with other subsequent analysis units, providing great potential in the process of analyzing sexual assault evidence as well as in other areas requiring cell sorting.

The application of alkaline lysis and pressure cycling technology in the differential extraction of DNA from sperm and epithelial cells recovered from cotton swabs.

This study reports the development of a two-step protocol using pressure cycling technology (PCT) and alkaline lysis for differential extraction of DNA from mixtures of sperm and vaginal epithelial cells recovered from cotton swabs. In controlled experiments, in which equal quantities of sperm and female epithelial cells were added to cotton swabs, 5 min of pressure pulsing in the presence of 0.4 M NaOH resulted in 104 ± 6% recovery of female epithelial DNA present on the swab. Following the pressure treatment, exposing the swabs to a second 5-min alkaline treatment at 95 °C without pressure resulted in the selective recovery of 69 ± 6% of the sperm DNA. The recovery of the vaginal epithelia and sperm DNA was optimized by examining the effect of sodium hydroxide concentration, incubation temperature, and time. Following the alkaline lysis steps, the samples were neutralized with 2 M Tris (pH 7.5) and purified with phenol-chloroform-isoamyl alcohol to permit downstream analysis. The total processing time to remove both fractions from the swab was less than 20 min. Short tandem repeat (STR) analysis of these fractions obtained from PCT treatment and alkaline lysis generated clean profiles of female epithelial DNA and male sperm DNA for 1:1 mixtures of female and male cells and predominant male profiles for mixtures up to 5:1 female to male cells. By reducing the time and increasing the recovery of DNA from cotton swabs, this new method presents a novel and potentially useful procedure for forensic differential extractions.

Spermatocele in a South African Boerboel Dog

A 2-year-old intact male South African Boerboel presented for semen cryopreservation and was discovered to be azoospermic. The dog had excellent libido and had sired litters within 6 months, so a further investigation of why his collection lacked sperm was warranted. On further examination of his scrotal contents, his right epididymis had an enlarged area with a hard texture. Ultrasonography revealed that the enlarged area of the right epididymis was fluid filled. A sample of the fluid was aspirated for aerobic culture. No bacteria showed growth. Although the culture was negative, it was suspected that this dog had an epididymitis or epididymal abscess, and treatment with enrofloxacin at 10mg/kg orally was initiated for 4 weeks. The abnormal texture and fluid-filled cavity in the right epididymis persisted, despite antibiotic therapy. Cytology of a repeat aspiration of the fluid-filled area after antibiotic therapy revealed a mixture of red blood cells and sperm. Owing to the potential for blood-testis barrier disruption, a unilateral orchiectomy of the right testicle was performed, as an attempt to protect future sperm production of the remaining testicle. A spermatocele was confirmed on histopathology. After another month, an excellent-quality semen sample was collected, with 90% progressive motility, good concentration, and few morphologic abnormalities. A subsequent collection was acquired and was successfully cryopreserved for future breeding. In dogs with spermatoceles, semen quality can be preserved with aggressive treatment to remove the affected testicle. The disruption of the blood-testis barrier in spermatoceles may result in antisperm antibody production and eventual infertility; however, cryopreservation can result in long-term options for owners seeking to continue using an animal in their breeding program.

A new strategy for sperm isolation and STR typing from multi-donor sperm mixtures.

Mixed semen stains from multiple contributors are challenging samples in sexual assault casework, and it is crucial to obtain the DNA profiles of different donors to allow the evidence to play an important role in investigations and judicial proceedings. Current standard procedures, including preferential lysis, are incapable of separating single-source sperm from multiple male donors. Mixed profiles are often obtained and may not directly exclude or identify suspects. In this case, computational methods for mixture interpretation are often used, which rely on different types of calculation models. Here, we explored a new strategy for sperm cell isolation and detection from mixtures. It is a direct way to obtain genotypes of different sperm donors compared to computation-based mixture interpretation. Laser capture microdissection (LCM) and low volume-PCR (LV-PCR) were used for single sperm isolation and detection. The platform was sensitive; profiling of a single sperm cell generated a minimum of 13-16 loci in 73.1% of Y short tandem repeat (Y-STR) assays. A new Y-STR and autosomal STR multiplex system (YA-STR) were optimized by the combination of the Y-STR locus and 10 autosomal STR (auto STR) loci. The Y-STR locus acted as a tag to discriminate profile groups from different donors. Subsequently, consensus auto STR profiles of various persons could be received. The accuracy and availability of this method were evaluated on a three-donor semen mixture and found to be effective for the resolution of a multi-donor sperm mixture.

Proteolytic system of the sperm of Apis mellifera drones

During many insemination interventions semen coagulates already within the insemination needle, which considerably lengthens the duration of inseminating a single queen bee. Considering this, the authors decided to determine the type and activity of proteases and their inhibitors in normal and coagulated sperm. The samples were collected from mature and old drones. The sperm proteins were isolated in 1% Triton X-100. The samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor activity detection by means of the modified Felicioli method. The mixing of non-coagulated semen from different drones increased protein concentration. The activities of proteases were decreased in normal sperm samples as compared with a corresponding rise in the sperm mixture from many drones. The non-coagulated sperm samples were found to contain aspartic and serine proteases. Additionally, thiolic and metallic proteases were also found in the coagulated sperm samples. There was a rise in protease inhibitor activity at pH 3.0 and 12.0, and a fall at pH 7.0 after mixing the sperm samples collected from numerous drones. Oscillation in these activities stemmed from sperm coagulation.

Does the order of copulation matter? Experimental paternity analyses in the earthworm Hormogaster elisae (Annelida: Hormogastridae)

Multiple mating and sperm storage organs allow postcopulatory sexual selection to occur. This is the first time that a paternity test has been performed in an earthworm species. Microsatellite markers are used in Hormogaster elisae to trace paternity for multiple sires. Multiple paternity was detected in some cases, showing sperm mixture even within one cocoon, suggesting the eventual mixture of the allosperm within the spermathecae. Order of copulation influences paternity, the first (P1) and third partner (P3) being the most successful. Interestingly the second partner (P2) almost lacks paternity. The most plausible hypothesis suggests the existence of sperm displacement by flushing out older sperm once the spermathecae are full, which seems to occur when they contain sperm from two partners and P3 donates. At that moment sperm is still stratified thus removing sperm from P2. Afterwards sperm from P1 and P3 would be mixed provoking an equivalent paternity. Given the endogeic nature of the earthworm, copulation was not observed, making the lack of copulation with P2 (due to the need of a recovery time) or copulation without sperm transfer (due to low sperm production rate) possible alternative hypotheses. The weight of the earthworms is related to the number of cocoons they produce but not to their viability, which in all the cases was low, probably due to laboratory conditions. The sperm was kept viable inside the spermathecae for a maximum of two years and three months, suggesting a very effective nourishment system in the epithelium of the storage structures.

No effect of sperm interactions or egg homogenate on sperm velocity in the blue mussel,Mytilus edulis(Bivalvia: Mytilidae)

We investigated the possible effects of sperm interactions and homogenized eggs on sperm velocity in blue mussels ( Mytilus edulis L., 1758) using computer-assisted sperm analysis. To test whether sperm competition results in an increase in sperm velocity, using seven pairs of males, we compared the mean curvilinear and average path velocities of sperm from two males measured separately with the corresponding values from a mixture of sperm from the same two males. To test whether the presence of eggs results in an increase in sperm velocity, we compared curvilinear and average path velocities from 11 individual males with the corresponding measures from the same 11 sperm samples mixed with aliquots of homogenized eggs. Neither experimental treatment resulted in an increase in sperm velocity. We interpret these results as consistent with the hypothesis that mussel sperm have been selected to immediately begin swimming at an optimal initial velocity that is adaptive for the particular environment in which they are located. Critical factors affecting the evolution of sperm velocity for broadcast spawning, external fertilizers such as M. edulis likely include population density and intraspecific spawning synchronicity. As has been suggested by others, the importance of sperm limitation (i.e., having much less than 100% of eggs being fertilized in the wild) may be as important an evolutionary driving force in broadcast spawning invertebrates as sperm competition is in internally or directly fertilized species.

Altering The Primal Environment: Health Effects Associated With Assisted Reproductive Technologies

When Darine El-Chaar began her residency in obstetrics and gynecology at the University of Ottawa five years ago, she grew curious about the potential health repercussions of assisted reproductive technologies (ART), the catchall term for procedures used to help couples artificially conceive a child. ART involves surgically removing eggs from a woman’s ovaries, combining them with sperm in the laboratory, and returning them to the womb.1 Women undergoing ART take “fertility drugs” such as clomiphene citrate and gonadotropins to stimulate the production of many eggs rather than the single egg that would normally grow during their monthly menstrual cycle.2 El-Chaar wondered about the influence that ART procedures, as well as the underlying infertility itself, might have on the health of children conceived. She is one of many researchers working to answer the fundamental question of whether introducing fertility drugs and manipulating eggs and sperm in a laboratory setting—in essence, altering the primal environment—sets the stage for adverse health effects in children. Artist’s rendering of intracytoplasmic sperm injection, in which a single sperm is inserted into an egg. Although some research indicates such a risk exists, there haven’t been enough large-scale studies to ascertain if the potential effects are severe enough over the long term to deter couples from seeking infertility treatments. Meanwhile, some findings are pointing the way toward possible refinements to improve health outcomes for ART babies.