Semen sexing and its impact on fertility and genetic gain in cattle.

Semen sexing is among one of the most remarkable inventions of the past few decades in the field of reproductive biotechnology. The urge to produce offspring of a desired sex has remained since traditional times. Researchers have tried many methods for accurate semen sexing, but only the flow cytometry method has proved to be effective for commercial utilization. However, there were always concerns about the effects of sexed semen, especially on fertility and the rate of genetic gain. Some concerns were genuine because of factors such as low semen dosage in sexed semen straws and damage to sperm during the sorting process. Various researchers have conducted numerous studies to find out the effect of sexed semen on fertility and, in this article, we reflect on their findings. Initially, there were comparatively much lower conception rates (∼70% of conventional semen) but, with refinement in technology, this gap is bridging and the use of sexed semen will increase over time. Concerning genetic gain with use of sexed semen, a positive effect on rate of genetic progress with the use of sexed semen has been observed based on various simulation studies, although there has been a mild increase in inbreeding.

Biochemical Features of X or Y Chromosome-Bearing Spermatozoa for Sperm Sexing.

This review presents information on biochemical features of spermatozoa bearing X or Y chromosome, enabling production of a sperm fraction with pre-defined sex chromosome. The almost only technology currently used for such separation (called sexing) is based on the fluorescence-activated cell sorting of sperm depending on DNA content. In addition to the applied aspects, this technology made it possible to analyze properties of the isolated populations of spermatozoa bearing X or Y chromosome. In recent years, existence of the differences between these populations at the transcriptome and proteome level have been reported in a number of studies. It is noteworthy that these differences are primarily related to the energy metabolism and flagellar structural proteins. New methods of sperm enrichment with X or Y chromosome cells are based on the differences in motility between the spermatozoa with different sex chromosomes. Sperm sexing is a part of the widespread protocol of artificial insemination of cows with cryopreserved semen, it allows to increase proportion of the offspring with the required sex. In addition, advances in the separation of X and Y spermatozoa may allow this approach to be applied in clinical practice to avoid sex-linked diseases.

A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters.

Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion-"X-shredding" and "X-poisoning"-and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Our results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when we directed Cas9 cleavage to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species.

Evaluation of sexed semen-based artificial insemination in Tharparkar cattle under organized farm condition.

Sexed semen facilitates additional female calf production for the expansion of a herd at a faster rate and also curtails the surplus production of unwanted male calves. A study was conducted to evaluate the performance of sexed semen in indigenous Tharparkar cows based on 114 artificial inseminations (AI) performed at natural oestrus using two protocols i.e., single AI (n = 48) and double AI (n = 66). Overall, the first service conception rate (CR) was significantly higher in double (53.0%) than single (33.3%) AI protocol. The odds ratio of conception rate in double AI was 2.26 (χ2 = 4.4, df = 1, p = .04) with respect to single AI. The time that elapsed since the detection of oestrus to insemination was also analysed. In a single AI protocol, the CR was higher (p < .05) at 16 h (54.6%) than insemination at 8 h (27.0%) following the onset of oestrus. Yet, the CR using double AI protocol did not differ (p = .73) significantly when AIs were performed either at 8 h and 24 h (51.9%) or 16 h and 24 h (57.1%) post onset of oestrus. Besides, like the single AI protocol, the parity of the animals also influenced the CR, being higher in heifers (n = 22) than those of parous (n = 92) cows (72.73 vs. 40.43%, χ2 = 7.48, df = 1, p = .006) in the present study. The odds ratio of conception in heifers was 3.93 with respect to parous cows. Overall, the birth of female calf was 91.7%. In conclusion, the present study indicates a future promise of the sexed semen for the production of more female offspring from Tharparkar cattle.

Short-term storage of semen samples in acidic extender increases the proportion of females in pigs.

BACKGROUND: Sex preselection is a desired goal of the animal industry to improve production efficiency, depending on industry demand. In the porcine industry, there is a general preference for pork from female and surgically castrated male pigs. Therefore, the birth of more females than males in a litter leads to economic benefits and improved animal welfare in the pig production industry. Our previous study suggested that the porcine semen extender (BTS) adjusted to pH 6.2 maximises the differences in viability between X-chromosome-bearing (X) spermatozoa and Y-chromosome-bearing (Y) spermatozoa without affecting sperm's functional parameters. In this study we aimed to evaluate whether the pH 6.2 extender is applicable at the farm level for increasing the number of female piglets without a decline in spermatozoa fertility. Artificial insemination (AI) was carried out with spermatozoa stored at pH 6.2 and pH 7.2 (original BTS) at day 1 and day 2 of storage. Next, the functional parameters of the spermatozoa, litter size, farrowing rate, and female-to-male ratio of offspring were determined. RESULTS: Although sperm motility decreased significantly after 2 d of storage, the viability of spermatozoa was preserved at pH 6.2 for 3 d. There was no significant difference in the farrowing rate and average litter size between the group inseminated with the spermatozoa stored in (pH 7.2) and that inseminated with spermatozoa stored in acidic BTS. The percentage of female piglets was approximately 1.5-fold higher in sows inseminated on day 1 in the pH 6.2 than in the pH 7.2 group. Furthermore, although there was no significant difference in the female-to-male ratio, the percentage of female piglets born was slightly higher in the pH 6.2 group than in the pH 7.2 group on day 2. CONCLUSIONS: The method optimised in our study is simple, economical, and may enhance the number of female births without any decline in spermatozoa fertility.

Disparities among infertility patients regarding genetic carrier screening, sex selection, and gene editing.

PURPOSE: The purpose of this study is to evaluate the perspectives of infertility patients regarding genetic carrier screening, embryo sex selection, embryo research, and gene editing. METHODS: An anonymous 32-question survey was distributed electronically to all patients who seen at a single academic fertility center for at least one visit between June 2018 and September 2019. Survey questions evaluated patient perspectives on genetic carrier screening, embryo sex selection, embryo research, and gene editing. RESULTS: There were 1460 survey responses (32.0% response rate). There were significant differences in the proportion of respondents receiving genetic carrier screening between racial groups, 73.1% of White, 45.5% of Black, 49.4% of Hispanic, and 62.8% of Asian respondents. The likelihood of having genetic carrier screening was also significantly influenced by respondent income, insurance status, and religion. Religion significantly influenced the acceptance of embryonic research and embryonic sex selection. While only 8.9% felt that genetically modifying embryos for physical traits should be allowed, 74.1% felt that genetic modification to correct disease should be allowed. CONCLUSION: Racial, religious, and socioeconomic factors significantly impacted respondents' likelihood to have genetic carrier screening and views on embryo sex selection, embryo research, and gene editing. These findings highlight the importance of tailoring genetic counseling to the individual, acknowledging individual and cultural differences in agreement with genetic testing and emerging genetic therapies.

Comparison of pregnancy per AI of heifers inseminated with sex-sorted or conventional semen after oestrus detection or timed artificial insemination.

The objective of the study was to compare the fertility after using sex-sorted or conventional semen either with oestrus detection (EST) or timed artificial insemination (TAI) in Holstein heifers. Holstein heifers were randomly assigned to one of the following treatments in a 2 × 2 factorial design. Heifers in the EST group were inseminated with sex-sorted (n = 114) or conventional semen (n = 100) after spontaneous or induced oestrus. Heifers in the TAI, subjected to the 5-day Cosynch+Progesterone protocol (GnRH+P4 insertion-5d-PGF2α +P4 removal-1d-PGF2α -2d-GnRH+TAI), were inseminated with sex-sorted (n = 113) or conventional semen (n = 88). Statistical analyses were performed using PROC GLIMMIX procedure of SAS 9.4 (SAS Institute Inc., Cary, NC). Overall P/AI was 60.7% for EST and 54.2% for TAI regardless of types of semen and 68.1% for conventional and 48.9% for sex-sorted semen regardless of insemination strategies. Fertility of heifers inseminated with either sex-sorted (53.5%; 44.2%) or conventional (69.0%; 67.0%) semen did not differ between EST and TAI respectively. Besides, the interaction between the semen type and the insemination strategy was not significant for P/AI. The embryonic loss was significantly greater with sex-sorted semen (17.1%) compared to conventional semen (1.6%). There was no sire effect with sex-sorted semen on P/AI (52.6% vs. 46.2%) and embryonic loss (16.4% vs. 18.0%). As expected, sex-sorted semen resulted in more female calves (89.8% vs. 51.6%) than conventional semen. Thus, sex-sorted semen can be used with 5-day Cosynch+Progesterone protocol to eliminate the inadequate oestrus detection and to increase female calves born in dairy heifers.

Production of monoclonal antibody against recombinant bovine sex-determining region Y (SRY) and their preferential binding to Y chromosome-bearing sperm.

Separation of X and Y chromosome-bearing sperm is an appropriate method for the selection of desired sex of offspring to increase the profit in livestock industries. The purpose of this study was the production of a monoclonal antibody against recombinant bovine sex-determining region Y protein for separation Y sperm. The hybridoma cells from splenocytes of immunized female's balb/C mice and Sp2/0 cells were made. The binding affinity of our monoclonal antibody (mAbSRY2) was compared with mouse monoclonal SRY-15. The Western blot method indicated that mAbSRY2 successfully detected the rbSRY protein. The specificity and sensitivity of mAbSRY2 is comparable to SRY-15 commercially ones. The SRY gene in 100% of bull semen contains the Y chromosome that had the strongest binding affinity to mAbSRY2 was synthesized. In other words, the binding affinity of semen contains the X sperms near the negative control. In general, this immunological method can help to separate X from Y sperms. However, the mAbSRY2 is bind to Y-bearing sexed sperm, but in the future; the sexed sperms need to apply in farms.

Recovery of sperms bearing X chromosomes with different concentrations of magnetic nanoparticles in ram.

Pre-conceptual sex selection is still a highly debatable process whereby X and Y chromosome bearing spermatozoa are isolated before oocyte fertilization. Recently, magnetic nanoparticles (MNP) have been used to determine X and Y chromosomes bearing spermatozoa as a result of searching for a cheap, highly efficient method using non-toxic materials. This study aimed to recover the sperm bearing X chromosomes in ram with different concentrations of MNP and then evaluate the success of this method using polymerase chain reaction (PCR). Ram sperms were divided into four groups, treated with 0 (control), 50, 100 and 200 µg/ml MNP, respectively. MNP was used to restore sperm cells bearing X chromosomes. Upon recovery, the PCR was performed to identify the X and Y sperms, Methyl ThiazoleTetrazolium (MTT), to assess MNP toxicity and sperm viability and acridine orange (AO) to evaluate sperm DNA integrity. The results of PCR revealed that the treatment of spermatozoa- bearing X chromosomes with 50 µg/ml MNP had the highest effects on the recovery of X sperm rather than the other concentrations of MNP. However, the concentrations of MNP did not have any toxic effects on spermatozoa, sperm viability and, DNA integrity, but the high concentration of MNP (200 µg/ml) significantly reduced DNA integrity. According to MTT and AO results, the concentrations of MNP used in this study had no toxic effects on spermatozoa and did not reduce the sperm viability and DNA integrity, except that 200 µg/ml MNP significantly reduced DNA integrity.

Production of sexed bovine embryos in vitro can be improved by selection of sperm treatment and co-culture system.

The study investigated the effects of sperm sorting, capacitation treatment and co-cultivation on sexed bovine in vitro embryo production. The effect of treatment and co-culture on production of embryos of the preferred sex from unsorted sperm was also studied. Sperm from five breeding bulls was used for fertilization of mature oocytes as follows: Experiment 1, sorted and unsorted sperm (bulls A-E) treated only with heparin in standard co-cultures; Experiment 2, sorted sperm (bulls A-E) treated with heparin-PHE (penicillamine, hypotaurine, and epinephrine) or heparin-caffeine in drop co-cultures; and Experiment 3, unsorted sperm (bull E) treated with either heparin-PHE or heparin-caffeine in both standard and drop co-cultures. In all bulls, treatment with heparin resulted in significantly (p < .05) reduced cleavage and blastocyst rates from sorted sperm, as compared with those from unsorted sperm. In bulls A, B, D and E, treatment of sorted sperm with heparin-PHE in drops significantly increased the blastocyst rate (p < .05). In unsorted sperm of bull E, heparin-PHE treatment in drops resulted in the XX/XY sex ratio inverse to that obtained by heparin-caffeine treatment in standard co-cultures (32.3%/67.7% and 66.7%/33.3%, respectively). In conclusion, the treatment of sorted sperm with heparin-PHE in modified drop co-cultures can be recommended for production of in vitro sexed embryos. The use of unsorted sperm for production of embryos of the preferred sex by selected capacitation treatment and co-culture can be the method of choice in bulls with low IVF yields from sorted sperm.

New technique of sex preselection for increasing female ratio in boar sperm model.

In this study, we tried to optimize the porcine semen extender conditions to maximize the differences between live X chromosome-bearing (X) spermatozoa and to Y chromosome-bearing (Y) spermatozoa without a decline in the fertility rate at different pH conditions during storage. We observed the viability of X and Y boar spermatozoa in acidic (pH 6.2), original (pH 7.2), and alkaline condition (pH 8.2) for 5 days to investigate the effect of storage conditions on the X to Y spermatozoa ratio. The functional parameters of spermatozoa were also examined to evaluate sperm quality. Sperm motility was preserved at pH 7.2 and pH 6.2 for 3 days, while sperm motility at pH 8.2 decreased significantly after 2 days. Non-capacitated spermatozoa increased while capacitated spermatozoa decreased during storage. Sperm viability decreased significantly duration-dependent under all pH conditions, but there was no significant difference during storage at pH 6.2 and 7.2. The X: Y ratio of live spermatozoa in acidic condition was maximized (1.2:1) without affecting the sperm function and fertility-related protein expression after 2 days compared to original conditions. Moreover, insemination of sows using acidic extender increased the number of female pups on days 1 and 2 of preservation. These results indicate that the production of female offspring may increase when acidic BTS is used for 2 days without affecting the success rate of AI. Above all, this method is simple and economical compared to other methods.

A dual colour FISH method for routine validation of sexed Bos taurus semen.

BACKGROUND: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. RESULTS: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). CONCLUSIONS: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.

Comparative evaluation of the cost and efficiency of four types of sexing methods for the production of dairy female calves.

This study was conducted to evaluate and compare the economic benefits of different embryo sexing methods, based on the cost per female dairy calf produced. Female calves were produced from four kinds of female embryos: (1) those collected from superstimulated donors at 7-8 days after artificial insemination (AI) with X-sorted semen; (2) those sex-determined by loop-mediated isothermal amplification assay of a biopsy sample of embryos collected from superstimulated donors after AI with conventional unsorted semen; (3) those obtained by invitro embryo production (IVEP), using X-sorted semen and in vitro-matured oocytes collected from donors by ovum pick-up (OPU); and (4) those obtained by IVEP, using X-sorted semen and oocytes collected by OPU after dominant follicle ablation and follicle growth stimulation of the donors. The respective productivities of female calves per technical service and the total production cost per female calf of each sexing method were compared. The production cost per female calf (66,537 JPY), as calculated from the number of female calves per service (1.30), pregnancy rate of transfer (42.9%), rate of female calves obtained (92.9%), and total cost of the method (56,643 JPY plus embryo transfer fee), was less for IVEP with X-sorted semen and follicular growth-stimulated (FGS) oocytes than for the other groups (P < 0.05). The results demonstrate that embryo production with X-sorted semen and FGS oocytes provides a more efficient method for producing female calves than the other embryo sexing methods.

Global sex selection techniques for family planning: a narrative review.

OBJECTIVE: To document the varying methods of sex selection, both primitive (traditional) as well as advanced forms available around the world. CONTEXT: With the increasing desire of couples to choose the gender of their offspring, scientific sex-selection methods and techniques have evolved over time; unfortunately, the medical and social consequences have remained poorly emphasised. METHODS: We searched electronic search engines and grey literature that included research articles from journals, books, websites and news articles in English until August 2016. We comprehensively compiled the findings such as underlying principles, time of use in relation to conception and others. RESULTS: We classified the techniques into natural methods that rely on physiological conditions and artificial methods, including manipulation of seminal fluid for sex selection. Natural methods include Shettles technique, Whelan Method, Billings Ovulation Method, pre-conception diet, and gender selection kits such as GenSelect and Smart Stork, which rely on timing of intercourse, the vaginal environment, a selective diet and nutraceuticals. More advanced and artificial methods include sperm sorting or Ericsson's method, Microsort, Preimplantation Genetic Diagnosis and Urobiologics PreGender test. The markets for these techniques are prevalent in India where the birth of a son is desired. There is also widespread use of indigenous medicines for sex selection. The review reports side effects such as vaginal infections, hyperstimulation syndrome, multiple pregnancies, birth defects and stillbirths. CONCLUSION: We conclude that sex-selection practices need urgent intervention in view of the social harm, unwarranted gender bias, and diversion of resources from genuine medical need.

In vitro characterisation of fresh and frozen sex-sorted bull spermatozoa.

This study sought to compare the in vitro characteristics of fresh and frozen non-sorted (NS) and sex-sorted (SS) bull spermatozoa. Experiment 1: Holstein-Friesian ejaculates (n=10 bulls) were split across four treatments and processed: (1) NS fresh at 3×106 spermatozoa, (2) X-SS frozen at 2×106 spermatozoa, (3) X-SS fresh at 2×106 spermatozoa and (4) X-SS fresh at 1×106 spermatozoa. NS frozen controls of 20×106 spermatozoa per straw were sourced from previously frozen ejaculates (n=3 bulls). Experiment 2: Aberdeen Angus ejaculates (n=4 bulls) were split across four treatments and processed as: (1) NS fresh 3×106 spermatozoa, (2) Y-SS fresh at 1×106 spermatozoa, (3) Y-SS fresh at 2×106 spermatozoa and (4) X-SS fresh at 2×106 spermatozoa. Controls were sourced as per Experiment 1. In vitro assessments for progressive linear motility, acrosomal status and oxidative stress were carried out on Days 1, 2 and 3 after sorting (Day 0=day of sorting. In both experiments SS fresh treatments had higher levels of agglutination in comparison to the NS fresh (P<0.001), NS frozen treatments had the greatest PLM (P<0.05) and NS spermatozoa exhibited higher levels of superoxide anion production compared with SS spermatozoa (P<0.05). Experiment 1 found both fresh and frozen SS treatments had higher levels of viable acrosome-intact spermatozoa compared with the NS frozen treatments (P<0.01).

Effects on glycocalyx structures of frozen-thawed bovine sperm induced by flow cytometry and artificial capacitation.

Sperm sorting by flow cytometry is a useful technology in the bovine industry, but the conception rates after artificial insemination using sex-sorted sperm are lower than when using the un-sorted sperm. In this study, we have investigated the causes for these low conception rates. We have focused on changes caused by flow cytometry to the glycocalyx, which forms the outermost surface of the sperm membrane. We have also evaluated the effects of capacitation on the glycocalyx since capacitation involves a redistribution of the sperm membrane that is vital for successful fertilization and conception. Lectin histochemistry was used to visualize the structure of the sperm glycocalyx. Lectin-staining sites were examined in non-treated sperm, sex-sorted sperm, and capacitated sperm. We have detected six different staining patterns related to different labeling regions of the sperm. Phaseolus vulgaris-erythroagglutinin (PHA-E) lectin-staining patterns of non-treated sperm were very different from those observed for sex-sorted sperm or capacitated sperm, suggesting that both, sex sorting by flow cytometry and the capacitation process affected the glycocalyx structures in the sperm. In addition, the total tyrosine-phosphorylation level in sex-sorted sperm was significantly higher than that in the non-treated sperm. Therefore, we concluded that the unexpected capacitation of bovine sperm during flow cytometry is associated with changes in the glycocalyx. Since premature capacitation leads to low conception rates, this unexpected capacitation could be a cause of low conception rates after artificial insemination using sex-sorted sperm.

Postnatal phenotype of dairy cows is altered by in vitro embryo production using reverse X-sorted semen.

Abnormal fetuses, neonates, and adult offspring derived by assisted reproductive technologies have been reported in humans and mice and have been associated with increased likelihood of certain adult diseases. To test the hypothesis that bovine females derived by assisted reproductive technologies have altered postnatal growth and adult function, a retrospective cohort study evaluated survival, growth, and production traits of offspring derived by in vitro embryo production (IVP) with conventional (IVP-conv) or reverse X-sorted semen (IVP-sexed), multiple ovulation and embryo transfer, and artificial insemination (AI) in a large dairy herd. Live calves produced by IVP were born slightly heavier compared with AI calves. In addition, IVP-sexed calves had a higher cumulative mortality from 90 to 180 d of age compared with AI offspring. Mortality of IVP-conv and multiple ovulation and embryo transfer offspring was intermediate and not different from AI or IVP-sexed offspring. The altered phenotype of offspring from IVP-sexed extended to adult milk production. Cows derived by IVP-sexed produced less milk, fat, and protein in their first lactation compared with dairy cows derived by AI. Additionally, females born to nulliparous dams had a distinct postnatal phenotype compared with offspring from parous dams even when data were restricted to offspring of surrogate females. In conclusion, procedures associated with in vitro production of embryos involving use of reverse-sorted spermatozoa for fertilization result in an alteration of embryonic programming that persists postnatally and causes an effect on milk production in adulthood. Thus, some benefits of reverse-sorted semen for genetic improvement may be offset by adverse programming events.

Flow cytometric sex sorting affects CD4 membrane distribution and binding of exogenous DNA on bovine sperm cells.

Bovine sex-sorted sperm have been commercialized and successfully used for the production of transgenic embryos of the desired sex through the sperm-mediated gene transfer (SMGT) technique. However, sex-sorted sperm show a reduced ability to internalize exogenous DNA. The interaction between sperm cells and the exogenous DNA has been reported in other species to be a CD4-like molecule-dependent process. The flow cytometry-based sex-sorting process subjects the spermatozoa to different stresses causing changes in the cell membrane. The aim of this study was to elucidate the relationship between the redistribution of CD4-like molecules and binding of exogenous DNA to sex-sorted bovine sperm. In the first set of experiments, the membrane phospholipid disorder and the redistribution of the CD4 were evaluated. The second set of experiments was conducted to investigate the effect of CD4 redistribution on the mechanism of binding of exogenous DNA to sperm cells and the efficiency of lipofection in sex-sorted bovine sperm. Sex-sorting procedure increased the membrane phospholipid disorder and induced the redistribution of CD4-like molecules. Both X-sorted and Y-sorted sperm had decreased DNA bound to membrane in comparison with the unsorted sperm; however, the binding of the exogenous DNA was significantly increased with the addition of liposomes. Moreover, we demonstrated that the number of sperm-bound exogenous DNA was decreased when these cells were preincubated with anti-bovine CD4 monoclonal antibody, supporting our hypothesis that CD4-like molecules indeed play a crucial role in the process of exogenous DNA/bovine sperm cells interaction.

Causes and estimated incidences of sex-chromosome misdiagnosis in preimplantation genetic diagnosis of aneuploidy.

Preimplantation genetic diagnosis of aneuploidy (PGD-A) with comprehensive chromosome analysis has been known to improve pregnancy outcomes. Accuracy in detecting sex chromosomes becomes important when selecting against embryos at risk for sex-linked disorders. A total of 21,356 PGD-A cycles consisting of day-3 (cleavage) or day-5 (blastocyst) biopsies were received at the same laboratory for PGD-A via fluorescence in situ hybridization (FISH) or array comparative genome hybridization (aCGH) from multiple fertility centres. The misdiagnosis rates were 0.12% (Wilson 95% CI 0.05 to 0.25%) in day-3 FISH cycles, 0.48% (Wilson 95% CI 0.19 to 1.22%) in day-3 aCGH cycles and 0.0% (Wilson 95% CI 0 to 0.26) in day-5 aCGH cycles. Although rare, the likely causative biological event for true misdiagnosis is embryonic XX/XY mosaicism. Reanalysis of 1219 abnormal cleavage-stage research embryos revealed a 73% incidence of minor and major mosaicism. Only four (0.3%) embryos were found to be diploid and contained XX and XY cells that could potentially account for the misdiagnosis of sex. Our investigation identified errors leading to misdiagnosis and their attribution to specific events during PGD-A testing. The reported misdiagnosis rates suggest that PGD-A for sex determination is highly accurate, particularly when using aCGH applied to blastocyst biopsies.

Risk Factors for Stillbirth: Findings from a Population-Based Case-Control Study, Haryana, India.

BACKGROUND: Stillbirth is a prevalent adverse outcome of pregnancy in India despite efforts to improve care of women during pregnancy. Risk factors for stillbirths include sociodemographic factors, medical complications during pregnancy, intake of harmful drugs, and complications during delivery. The objective of the study was to examine the risk factors for stillbirth with a focus on sex selection drugs (SSDs). METHODS: A population-based case-control study was undertaken in Haryana. Cases of stillbirths were identified from the Maternal Infant Death Review System portal of Haryana state for the months of August-September 2014. A consecutive birth from the same geographical area as the case was selected as the control. The sample size was 325 per group. Mothers were interviewed using a validated tool. Bivariate analyses and logistic regression were conducted to examine the association between risk factors and stillbirth. Attributable risk proportions (ARP) and population attributable risk proportions (PARP) were estimated. RESULTS: The sociodemographic profiles of the cases and controls were similar. History of intake of SSDs [adjusted odds ratio (OR) 2.6, 95% confidence interval (CI) 1.5, 4.5] emerged as a risk factor. Other significant factors were preterm <37 weeks (OR 3.5, 95% CI 2.1, 6.0), history of previous stillbirths (OR 4.0, 95% CI 2.1, 7.8), and complications during labour (OR 3.3, 95% CI 2.1, 5.3). Estimates of the ARP and PARP for intake of SSDs were 0.60 (95% CI 0.32, 0.77) and 0.1 (95% CI -0.13, 0.28), respectively. CONCLUSIONS: SSDs could be attributed as a risk factor in a fifth of the cases of stillbirths. The number needed to harm for the use of SSDs in causing adverse effect of stillbirths was 5, suggesting thereby that for every five mothers exposed to SSDs, one would have stillbirth. Greater efforts are required to inform people about the harmful effects of SSD consumption during pregnancy.