N. Kanakas

Effects of paternal cigarette smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation in rats

Summary. We evaluated the effects of paternal smoking on testicular function, sperm fertilizing capacity, embryonic development, and blastocyst capacity for implantation. Rats of group A were exposed to cigarette smoke for 10 weeks. Rats of group B were exposed to the smoke of incense sticks for 10 weeks. Rats of group C served as a control group. Rats of group D were exposed to cigarette smoke for 7 weeks only. Experimental period was 10 weeks in all groups. At the end of the experimental period serum testosterone responses to human chorionic gonadotropin stimulation, andro‐gen‐binding protein activity in testicular cytosols, epididymal sperm motility, and oocyte fertilization rate, oocyte cleavage rate, and blastocyst development rate after in vitro fertilization (IVF) trials were significantly smaller in group A compared with groups B and C. In contrast, fertilization rate, cleavage rate, and blastocyst development rate after intracytoplasmic sperm injection (ICSI) procedures were not significantly different among groups A, B, C, and D. Both after IVF trials and ICSI techniques, the proportion of the alive offspring to the number of transferred oocytes was significantly smaller in group A than in groups B and C. Cigarette smoke‐exposure results in a secretory deficiency of Leydig and Sertoli cells leading to an impaired epididymal sperm maturation process and diminished capacity of spermatozoa to penetrate oocytes. In addition paternal cigarette smoke exposure affects the embryonic ability for implantation.

Effects of cotinine on sperm motility, membrane function, and fertilizing capacity in vitro.

Abstract We evaluated the effect of cotinine on sperm fertilizing capacity in vitro. Human spermatozoa were washed and re-suspended in medium containing albumin and various concentrations of cotinine (0, 100, 200, 400, or 800 ng/ml). After an 8-h incubation period, sperm motility, hypoosmotic swelling test (HOST) outcome, and the percentage of hyperactivated spermatozoa were assayed. Aliquots of spermatozoa were then processed for the zona-free hamster oocyte sperm penetration assay (SPA) or hamster ooplasmic injections. Spermatozoa exposed to concentrations of cotinine equal to 400 or 800 ng/ml demonstrated significantly smaller outcomes for all of the above with the exception of after hamster ooplasmic injections, where high cotinine concentrations did not affect sperm viability or sperm capacity to undergo decondensation and activate hamster oocytes. It appears that cotinine concentrations of 400 or 800 ng/ml exert a detrimental effect on sperm motility, membrane function, and the ability to undergo capacitation. In addition, the current findings suggest that smokers with a high seminal plasma cotinine concentration who participate in assisted reproduction programs may be treated with intracytoplasmic sperm injections (ICSI) rather than conventional in vitro fertilization (IVF) trials.

Erectile function and male reproduction in men with spinal cord injury: a review

Spinal cord injury (SCI) in men results in defects in erectile function, ejaculatory process and male reproductive potential. There are alterations in the capacity of men with SCI to achieve reflexogenic, psychogenic and nocturnal erections. The sexual function in different stages after SCI and the types of erections depend mainly on the completeness of the injury and the level of neurological damage. Furthermore, most of the SCI men demonstrate defects concerning the entrance of semen into the posterior urethra and the expulsion of the semen through the penile urethra and the urethral orifice. In addition, SCI men develop defects in the secretory function of the Leydig cells, Sertoli cells and the male accessory genital glands. The overall result is a decreased quality of the semen is recovered either with penile vibratory stimulation (PVS) or with electroejaculation. Nowadays the therapeutic andrological approach of SCI men focuses on achievement of erectile function, recovery of spermatozoa and assisted reproductive technology. The first line of therapy recommended for infertility in SCI men is collection of semen via PVS with concomitant evaluation of total motile sperm yields for assisted conception which may include intravaginal insemination, intrauterine insemination, or in vitro fertilisation/intracytoplasmic sperm injection. Patients failing PVS may be referred for electroejaculation or surgical sperm retrieval.

Role of testicular tissue telomerase assay for the prediction of the presence of testicular spermatozoa in azoospermic men with varicoceles, pre- and post-varicocelectomy.

Summary.  We evaluated the reproductive potential of frozen/thawed testicular spermatozoa of azoospermic men with left varicocele. The role of testicular tissue telomerase assay (TTA) in the prediction of the presence of testicular spermatozoa pre‐ and post‐varicocelectomy was investigated, as well. Therapeutic testicular biopsy and TTA were performed in 82 nonobstructed azoospermic (NOA) men with varicoceles. Testicular spermatozoa were found in 33 men and processed for cryopreservation. Oocytes were later recovered from the spouses of the latter azoospermic men with varicoceles and injected with frozen/thawed testicular spermatozoa. Among the 49 men who were negative for testicular spermatozoa, 22 men underwent subsequently subinguinal microsurgical varicocelectomy. A total of 198 mature oocytes were successfully injected and 101 were normally fertilized and subsequently cleaved. Transfer of these 101 embryos in 26 women resulted in nine full‐term pregnancies. Thirteen healthy babies were delivered. A cut‐off value of TTA of 39 TPG U μg−1 protein had an overall diagnostic accuracy equal to 90.2% to predict the presence of testicular spermatozoa pre‐varicocelectomy. Within the group of men who were negative for testicular spermatozoa a cut‐off value of TTA equal to 28 TPG U μg−1 protein (pre‐varicocelectomy) had a 84.2 % diagnostic accuracy to recognize the men who would become positive for either ejaculated or testicular spermatozoa post‐varicocelectomy. Testicular spermatozoa can be found in 40% of NOA men with left varicocele. Ooplasmic injections with frozen/thawed testicular spermatozoa have a role in the therapeutic management of non‐obstructive azoospermia associated with varicocele. Pre‐varicocelectomy, a TTA cut‐off value equal to 39 TPG U μg−1 protein has a 90.2% diagnostic accuracy to indicate the men positive/negative for testicular spermatozoa. In addition, pre‐varicocelectomy, a cut‐off value equal to 28 TPG U μg−1 protein has a 84.2% diagnostic accuracy to identify those men with varicoceles without testicular spermatozoa, who will become positive/negative for spermatozoa (either ejaculated or testicular) post‐varicocelectomy.

In vitro spermatogenesis as a method to bypass pre‐meiotic or post‐meiotic barriers blocking the spermatogenetic process: genetic and epigenetic implications in assisted reproductive technology

Pregnancies achieved by assisted reproduction technologies and particularly by ooplasmic injections of either in vivo or in vitro generated immature male germ cells are susceptible to genetic risks inherent to the male population treated with assisted reproduction and additional risks inherent to these innovative procedures. The documented, as well as the theoretical risks, are discussed in this review. These risks represent mainly the consequences of genetic abnormalities underlying male infertility and may become stimulators for the development of novel approaches and applications in the treatment of infertility. Recent data suggest that techniques employed for in vitro spermatogenesis, male somatic cell haploidisation, stem cell differentiation in vitro and assisted reproductive technology may also affect the epigenetic characteristics of the male gamete, the female gamete, or may have an impact on early embryogenesis. They may be also associated with an increased risk for genomic imprinting abnormalities. Production of haploid male gametes in vitro may not allow the male gamete to undergo all the genetic and epigenetic alterations that the male gamete normally undergoes during in vivo spermatogenesis.

The Effect of Valacyclovir Treatment on Natural Killer Cells of Infertile Women

Problem:  The aim of this study was to investigate the effect of valacyclovir treatment on natural killer (NK) cell concentration in the peripheral blood of infertile women.

Effects of primary testicular damage on sperm DNA oxidative status and embryonic and foetal development

We evaluated the development of embryos generated from the fertilisation of oocytes with spermatozoa isolated from animals with primary testicular damage (PTD). Embryos derived in vivo or in vitro from oocytes fertilised with spermatozoa produced by PTD rats that had undergone surgical treatment for the PTD (group A1), or PTD rats (group A2), or control rats (group B) were cultured and transferred to recipients. At the end of the experimental period, the fertilisation potential of each rat was assessed in vitro (IVF trials). Sperm 8‐oxodG/dG ratio (a marker of DNA oxidative status) was significantly larger in group A2 than in groups A1 and B. Blastocysts of the group A2 transferred to recipients demonstrated a significantly larger loss before implantation than transferred blastocysts of groups A1 or B. In addition, the proportion of implanted blastocysts that could not complete the intrauterine development was significantly larger in group A2 than in groups A1 and B. This study reveals a post‐fertilisation detrimental effect in animals with PTD on the capacity of oocytes (fertilised either in vitro or in vivo) to develop in vitro and implant after transferring them to recipients probably attributable to sperm DNA oxidative damage.

The role of ultrasonographically guided puncture of the human rete testis in the therapeutic management of nonobstructive azoospermia.

We attempted to characterize the cells collected from the rete testis via ultrasonographically guided puncture. Unilateral puncture of the rete testis was performed in nine men with obstructive azoospermia and 51 men with nonobstructive azoospermia. All the aspirated samples from the rete testis were observed via confocal scanning laser microscope and some of them after fluorescent in situ hybridization techniques. Then therapeutic testicular biopsy was performed in the punctured testis of each man. Spermatozoa were found in all rete testis samples and all biopsy samples from obstructed men. Twenty‐two nonobstructed men demonstrated absence of spermatozoa in biopsy samples. Twenty‐nine nonobstructed men showed spermatozoa in biopsy material and 24 of these men (82%) had demonstrated spermatozoa in rete testis samples. There were no significant differences in fertilization and cleavage rate between intracytoplasmic sperm injection trials using biopsy spermatozoa and rete testis spermatozoa both in obstructed and nonobstructed men. Considering that puncture of the rete testis does not reduce the volume of testicular parenchyma, is less invasive and apparently causes less detrimental effect on testicular vasculature than biopsy, puncture of rete testis is recommended as first line approach for the treatment of azoospermic men. If puncture is negative for spermatozoa in nonobstructed men, biopsy is indicated.

Post-fertilization effects of chronic renal failure in male rats.

We evaluated the potential for growth and intrauterine development of embryos generated from the fertilization of oocytes with spermatozoa recovered from animals with chronic renal failure (CRF). Group A included sham-operated rats (n = 28), group B1 involved CRF rats that had undergone erythropoietin plus bromocryptine treatment (n = 28), and group B2 included CRF rats that had received normal saline. Embryos derived from the in vitro fertilization of oocytes with spermatozoa recovered from rats of group A or group B1 or group B2 were transferred to female recipients. We induced CRF in a group of rats (group B; n = 56; the total kidney volume was reduced to one-sixth with two operations). One week after the second operation, the rats of group B were randomly divided into group B1 (they subsequently received bromocryptine plus erythropoietin) and group B2 (they received injections of saline). Nine weeks after the second operation, the fertility of each male rat was assessed by mating tests and in vitro fertilization of oocytes. The mean litter size was significantly smaller in the subpopulation of fertile animals in group B2 than in the fertile rats of group B1 and in the fertile rats of group B1 than in the fertile rats of group A. Per cent of transferred blastocysts that developed into alive offspring were significantly lower in group B2 than in group B1 and in group B1 than in group A. Epididymal spermatozoa demonstrated a significantly larger DNA-oxidative damage in group B2 than in group B1 and in group B1 than in group A. These findings demonstrate that sperm-DNA damage because of CRF development is accompanied by a defect in the development of embryos generated in vitro. We may suggest that bromocryptine and erythropoietin protecting sperm DNA from oxidative damage improve reproductive potential in rats with CRF.

Pre-decondensed sperm head injections into female pronuclei result in chromosomal mingling, zygotic cleavage, and adequate embryonic and fetal development up to delivery of healthy offspring: a novel method of assisted syngamy.

Summary.  Investigation of the developmental potential post‐injection of a pre‐decondensed or non‐pre‐decondensed sperm head into the female pronucleus of a pre‐activated oocyte. Rat pre‐activated oocytes were treated with intrapronuclear pre‐decondensed sperm head injections (IPSHI) (n = 133) or intrapronuclear non‐pre‐decondensed sperm head injections (INPSHI) (n = 138). All injected oocytes were transferred to pseudopregnant female recipients. Rat IPSHI techniques resulted in the delivery of five healthy offspring. Rat INPSHI techniques did not result in any pregnancies. Rat IPSHI techniques can result in delivery of healthy offspring. Successful performance of human IPSHI techniques might serve as a novel method to manage cases of intracytoplasmic sperm injection failure due to lack of development of male pronucleus or due to failure in pronuclei fusion.