Summary. Ejaculates (164) were obtained from 17 donors serving on an artificial insemination by donor panel. Semen analysis was performed before and after freezing by an integrated microcomputerized system employing the multiple-exposure photography (MEP) method. Sperm count, motility, velocity, motility index (MI; product of the sperm velocity and percentage of motile spermatozoa) and motile density (MD) were determined for each ejaculate. After the initial evaluation the ejaculates were frozen in liquid nitrogen, thawed 24 h later, and assessed for post-thaw motility, velocity, MI and MD. The mean ± s.e. sperm count and volume for this group of donors was 148 ± 4 × 106/ml and 3·1 ± 0·1 ml, respectively. Mean ± s.e. values obtained from the prefreeze analysis were: motility = 64 ± 1%, velocity = 30 ± 0·4μm/sec, MI = 19 ± 0·5 μm/sec and MD = 94 ± 3 × 106/ml. Post-thaw analysis revealed a significant reduction (P < 0·01 in all values measured. Motility was reduced to 27 ± 1%, MI was reduced to 5 ± 0·3 μm/sec, and MD was reduced to 33 ± 1 × 106/ml Velocity was the least affected by cryopreservation, being reduced to 21 ± 0·5 μm/sec (P < 0·01). Cryopreservation resulted in a marked shift in the frequency distribution of sperm motility and motility index towards subnormal values while in the majority of ejaculates velocity and motile density were maintained in the normal range. Significant differences were noted amongst donors in the percentage change of the various semen measures as a result of cryopreservation. When within-subject coefficients of variation were calculated, velocity was the least variable parameter. These results indicate that, while cryopreservation results in significant reductions in the number of motile spermatozoa in the ejaculate, the velocity is only marginally reduced.
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B. A. Keel, B. W. Webster, and D. K. Roberts
E. A. McLaughlin, W. C. L. Ford, and M. G. R. Hull
Summary. The motility characteristics of washed spermatozoa from 50 normal ejaculates were measured by time-lapse photography, before and after cryopreservation. Plasma membrane integrity was assessed by the hypo-osmotic swelling test and with the supravital fluorescent dye bisbenzimide (H33258).
There was a marked decline in the percentage of progressively motile spermatozoa after cryopreservation, the extent varying widely among donors. Results were, however, consistent between different ejaculates from the same individual. The ability of spermatozoa to survive cryopreservation could not be predicted from the properties of the semen beforehand.
The mean velocity of the spermatozoa was significantly reduced after freezing, but the lateral head displacement was unaltered. There was a significant reduction in the proportion of spermatozoa with intact plasma membranes after cryopreservation and the results of the hypo-osmotic swelling test and H33258 tests correlated closely. There was no correlation between the declines in the percentage of motile spermatozoa, or intact spermatozoa and the sperm velocity.
We conclude that membrane rupture is not the sole cause of loss of motile spermatozoa during freezing and that the decrease in the proportion of motile spermatozoa is caused, at least in part, by a separate process from that responsible for the decrease in the average swimming speed of spermatozoa.
Keywords: spermatozoa; cryopreservation; motility; membranes; semen; donor insemination; human
A. A. Templeton, P. Van Look, R. E. Angell, R. J. Aitken, M. A. Lumsden, and D. T. Baird
Summary. Volunteer women requesting laparoscopic sterilization were subjected to a fixed schedule of ovulation induction and oocyte recovery. Follicle aspiration was carried out in four groups: those to whom hCG was not administered and 12, 24 or 36 h respectively after the administration of hCG. For each group oocytes were cultured in vitro for 42 h, 30 h, 18 h and 6 h respectively, before insemination with donor spermatozoa. Oocyte recovery rates improved with longer hCG-to-recovery intervals (36% with no hCG to 81% 36 h after hCG). Although there was a slight reduction in fertilization rates when oocytes were not exposed to hCG in the follicle, normal cleavage was noted in more than 50% of oocytes in all four groups. It therefore appears that the final maturation stages of the human oocyte are not dependent on the midcycle gonadotrophin surge, provided the oocyte is matured in vitro before insemination. However, it was also evident that the fertilization rates were reduced when oocytes were removed from less mature follicles, as reflected by high androstenedione/ oestradiol ratios.
E. A. McLaughlin, W. C. L. Ford, and M. G. R. Hull
Summary. The contribution of the toxicity of glycerol–egg yolk–citrate (GEYC) cryopreservative medium to the loss of function of human spermatozoa during cryopreservation was determined by investigating the effect of mixing semen with the medium on sperm motility. The percentage of progressively motile spermatozoa, velocity (μm s−1) and lateral head displacement (μm) (mean ± sem, n = 28) were 55 ± 4·1, 47 ± 2·7, 4·4 ± 0·2 and 32 ± 3·8, 40 ± 2·5, 3·6 ± 0·25 and 15 ± 2·5, 28 ± 1·1, 2·8 ± 0·15 in suspensions of washed spermatozoa prepared from fresh, GEYC-treated and frozen–thawed semen, respectively. The variables changed only slightly after incubation for 3 h. The toxicity of GEYC did not vary significantly between samples which survived the complete freeze–thaw cycle well or very poorly. The toxicity of GEYC is responsible for about 50% of the loss of progressively motile spermatozoa during the complete cryopreservation process, but has little effect on the quality of motility. Susceptibility to GEYC does not explain observed differences in the ability of semen samples to survive freezing.
Keywords: cryopreservation; spermatozoa; motility; glycerol; donor insemination; human
Anamaria C Herta, Francesca Lolicato, and Johan E J Smitz
The currently available assisted reproduction techniques for fertility preservation (i.e. in vitro maturation (IVM) and in vitro fertilization) are insufficient as stand-alone procedures as only few reproductive cells can be conserved with these techniques. Oocytes in primordial follicles are well suited to survive the cryopreservation procedure and of use as valuable starting material for fertilization, on the condition that these could be grown up to fully matured oocytes. Our understanding of the biological mechanisms directing primordial follicle activation has increased over the last years and this knowledge has paved the way toward clinical applications. New multistep in vitro systems are making use of purified precursor cells and extracellular matrix components and by applying bio-printing technologies, an adequate follicular niche can be built. IVM of human oocytes is clinically applied in patients with polycystic ovary/polycystic ovary syndrome; related knowhow could become useful for fertility preservation and for patients with maturation failure and follicle-stimulating hormone resistance. The expectations from the research on human ovarian tissue and immature oocytes cultures, in combination with the improved vitrification methods, are high as these technologies can offer realistic potential for fertility preservation.
J. M. Sreenan and T. McDonagh
Summary. In Exp. 1, embryo survival rates of 45 and 47% were recorded after artificial insemination and ipsilateral transfer respectively. In Exp. 2, pregnancy rates of 62 and 60% were recorded after artificial insemination and contralateral transfer to inseminated recipients respectively. In this experiment the contralateral transferred embryo survival rate was 44%. Transferred embryo survival was lower overall when donors and recipients were out of phase by 1 day than when exactly synchronous.
K Hardy, C Wright, S Rice, M Tachataki, R Roberts, D Morgan, S Spanos, and D Taylor
The advent of human in vitro fertilization (IVF) over 30 years ago has made the oocyte and preimplantation embryo uniquely accessible. This accessibility has given rise to new micromanipulation techniques, such as intracytoplasmic sperm injection for treatment of male infertility, as well as embryo biopsy for preimplantation diagnosis of both genetic disease and aneuploidy, a major cause of early embryo demise and miscarriage. In the UK, average pregnancy rates after IVF and embryo transfer are < 25%, even after transfer of several embryos. Unfortunately, a third of these pregnancies involve multiple gestations. Research is currently focusing on methods to improve IVF success rates while reducing twin and triplet pregnancies and their associated increased morbidity and mortality. One approach is to develop screening methods to identify the most viable embryos, so that transfer of fewer healthy embryos will result in a higher proportion of singleton pregnancies. Screening methods include optimizing culture conditions for prolonged culture and selection of viable blastocysts for transfer, or embryo biopsy and aneuploidy screening. Assisted reproduction is also increasingly important in other branches of medicine: survival rates for cancer sufferers are improving continually and there is now a significant need for approaches to preserve fertility after sterilizing chemo-and radiotherapy treatment. Techniques for cryopreserving male and female gametes or gonadal tissue are being developed, although systems to grow and mature these gametes are in their infancy. Finally, there are also concerns regarding the safety of these new assisted reproductive technologies.
Darren K Griffin and Cagri Ogur
Designed to minimize chances of transferring genetically abnormal embryos, preimplantation genetic diagnosis (PGD) involves in vitro fertilization (IVF), embryo biopsy, diagnosis and selective embryo transfer. Preimplantation genetic testing for aneuploidy (PGT-A) aims to avoid miscarriage and live born trisomic offspring and to improve IVF success. Diagnostic approaches include fluorescence in situ hybridization (FISH) and more contemporary comprehensive chromosome screening (CCS) including array comparative genomic hybridization (aCGH), quantitative polymerase chain reaction (PCR), next-generation sequencing (NGS) and karyomapping. NGS has an improved dynamic range, and karyomapping can detect chromosomal and monogenic disorders simultaneously. Mosaicism (commonplace in human embryos) can arise by several mechanisms; those arising initially meiotically (but with a subsequent post-zygotic ‘trisomy rescue’ event) usually lead to adverse outcomes, whereas the extent to which mosaics that are initially chromosomally normal (but then arise purely post-zygotically) can lead to unaffected live births is uncertain. Polar body (PB) biopsy is the least common sampling method, having drawbacks including cost and inability to detect any paternal contribution. Historically, cleavage-stage (blastomere) biopsy has been the most popular; however, higher abnormality levels, mosaicism and potential for embryo damage have led to it being superseded by blastocyst (trophectoderm – TE) biopsy, which provides more cells for analysis. Improved biopsy, diagnosis and freeze-all strategies collectively have the potential to revolutionize PGT-A, and there is increasing evidence of their combined efficacy. Nonetheless, PGT-A continues to attract criticism, prompting questions of when we consider the evidence base sufficient to justify routine PGT-A? Basic biological research is essential to address unanswered questions concerning the chromosome complement of human embryos, and we thus entreat companies, governments and charities to fund more. This will benefit both IVF patients and prospective parents at risk of aneuploid offspring following natural conception. The aim of this review is to appraise the ‘state of the art’ in terms of PGT-A, including the controversial areas, and to suggest a practical ‘way forward’ in terms of future diagnosis and applied research.
JAN RABOCH and ZD. TOMáŠEK
The authors performed therapeutic donor insemination in 219 women. In 114 cases, 132 conceptions were obtained. The average number of inseminations for one conception was 3·8. More than half of the women in the `unsuccessful' subgroup did not exhaust the therapeutic possibilities of this treatment, i.e. aid extending over the period of five to six cycles. Seventeen losses in pregnancy, or within 10 days after delivery, correspond to less than 13%. One boy out of 108 living children (fifty boys and fifty-eight girls) had a developmental anomaly (penile hypospadias).
Alan H Handyside
The first pregnancies and live births following in vitro fertilisation (IVF) and preimplantation genetic testing (PGT), formerly known as preimplantation genetic diagnosis, were reported in 1990, almost 30 years ago, in several couples at risk of X-linked inherited conditions, which typically only affect boys inheriting the X chromosome with the affected gene from their carrier mothers. At that time, it was only possible to identify the sex of the embryo by amplifying a Y-linked repeat sequence in single cells biopsied at cleavage stages and avoid the transfer of males, half of which would be affected. The extensive publicity surrounding these cases and the perceived risk of using IVF and PGT for desirable characteristics not related to health, such as sex selection, led to the epithet of ‘designer babies’ which continues to resonate to this day. Here, I briefly reflect on how the technology of PGT has evolved over the decades and whether it deserves this reputation. With efficient methods for whole genome amplification and the genomic revolution, we now have highly accurate universal tests that combine marker-based diagnosis of almost any monogenic disorder with the detection of aneuploidy. PGT is now clinically well established and is likely to remain a valuable alternative for couples at risk of having affected children.