Surgical Vasectomy Research Articles

Naturally sterile Mus spretus hybrids are suitable for the generation of pseudopregnant embryo transfer recipients

For the preparation of embryo transfer recipients, surgically vasectomized mice are commonly used, generated by procedures associated with pain and discomfort. Sterile transgenic strains provide a nonsurgical replacement, but their maintenance requires breeding and genotyping procedures. We have previously reported the use of naturally sterile STUSB6F1 hybrids for the production of embryo transfer recipients and found the behavior of these recipients to be indistinguishable from those generated by vasectomized males. The method provides two substantial 3R impacts: refinement (when compared with surgical vasectomy) and reduction in breeding procedures (compared with sterile transgenic lines). Despite initial promise, the 3Rs impact of this innovation was limited by difficulties in breeding the parental STUS/Fore strain, which precluded the wider distribution of the sterile hybrid. The value of a 3R initiative is only as good as the uptake in the community. Here we, thus, select a different naturally sterile hybrid, generated from strains that are widely available: the B6SPRTF1 hybrid between C57BL/6J and Mus spretus. We first confirmed its sterility by sperm counting and testes weight and then trialed the recovery of cryopreserved embryos and germplasm within three UK facilities. Distribution of sperm for the generation of these hybrids by in vitro fertilization was found to be the most robust distribution method and avoided the need to maintain a live M. spretus colony. We then tested the suitability of B6SPRTF1 sterile hybrids for the generation of embryo transfer recipients at these same three UK facilities and found the hybrids to be suitable when compared with surgical vasectomized mice and a sterile transgenic strain. In conclusion, the potential 3Rs impact of this method was confirmed by the ease of distribution and the utility of sterile B6SPRTF1 hybrids at independent production facilities.

Replacement of surgical vasectomy through the use of wild-type sterile hybrids.

For the production and rederivation of mouse strains, pseudopregnant female mice are used for embryo transfer and serve as surrogate mothers to support embryo development to term. Vasectomized males are commonly used to render pseudopregnancy in females, generated by surgical procedures associated with considerable pain and discomfort. Genetically modified mouse strains with a sterility phenotype provide a non-surgical replacement and represent an important application of the 3Rs (Replacement, Reduction, Refinement). However, the maintenance of such genetically modified mouse strains requires extensive breeding and genotyping procedures, which are regulated procedures under national legislation. As an alternative, we have explored the use of sterile male hybrids that result when two wild-type mouse subspecies, Mus musculus musculus and Mus musculus domesticus, interbreed. We find the male STUSB6F1 hybrid, resulting from the mating of female STUS/Fore with male C57BL/6J, ideally suited and demonstrate that its performance for the production of oviduct and uterine transfer recipients is indistinguishable when compared to surgically vasectomized mice. The use of these sterile hybrids avoids the necessity for surgical procedures or the breeding of sterile genetically modified lines and can be generated by the simple mating of two wild-type laboratory strains-a non-regulated procedure. Furthermore, in contrast with the breeding of genetically sterile mice, all male offspring are sterile and suitable for the generation of pseudopregnancy, allowing their efficient production with minimal breeding pairs.

Pregnancy after vasectomy: surgical reversal or assisted reproduction?

Should we opt for surgical vasovasostomy or IVF/ICSI after a vasectomy? Both options reveal acceptable pregnancy rates though the time to pregnancy was significantly lower in the immediate IVF/ICSI group. About 7.4% of men regret their vasectomy and express a renewed child wish. The choice between surgical vasectomy reversal or ICSI remains difficult for patients and their fertility specialist. This study was a retrospective single-center cohort analysis of all males with a vasectomy in the past seeking treatment between 2006 and 2011 (n = 163). One group of patients opted for a reanastomosis procedure while the others opted for an immediate IVF/ICSI treatment. This included 99 males who underwent reanastomosis and 64 couples who immediately underwent ICSI treatment. All reanastomosis procedures were done by the same surgeon. ICSI was used in all cases where testicular sperm were extracted by fine needle aspiration (FNA) or testicular sperm extraction (TESE). The mean male age at vasectomy was 35.5 years and 44.4 years at reanastomosis. The mean (range) obstructive interval was 9.53 years (1-27). No significant differences were found between the two groups in female patient characteristics, such as age and parity. In the reversal group, the crude cumulative delivery rate (CDR) was 49.5%. However, in the 45 patients of this group who attempted to conceive spontaneously ('primary reanastomosis' pathway), the crude CDR was 40.0%. The remaining 54 patients (the 'switchers' pathway) who underwent a reversal procedure and later switched to ART, had a crude CDR of 57.4%. Of these, four patients opted for insemination, including two who later decided to switch to IVF/ICSI. The 64 patients who immediately underwent IVF/ICSI ('primary IVF/ICSI' pathway) had a crude CDR of 43.8% and an expected CDR of 51.6%. The difference in delivery rates between the primary reanastomosis group (40.0%) and the primary IVF/ICSI group (43.8%) was not statistically significant. Time to pregnancy was significantly shorter in the primary IVF/ICSI pathway, at 8.2 versus 16.3 months in the reanastomosis group. The study population was rather small. Furthermore, the study may be limited by the fact that the reason for the renewed child wish in most cases was a new relationship with another woman, a factor which may also play a role in the cause of infertility. Recanalisation of the vas seems to be a reasonable alternative for patients who do not wish to undergo immediate IVF/ICSI. In those who opt for ART immediately, the cumulative pregnancy rates seem comparable but the pregnancies occurred earlier. No funding was used for this study. There is no conflict of interest for this study. N/A.

Is Endoscopic Vasectomy Just a Dream: An ex vivo Study on Feasibility and Reliability of Endoluminal Occlusion of Porcine Vas Deferens

Introduction and Aim: Surgical vasectomy remains the gold standard for fertility control in men. Endoluminal occlusion of the seminal ducts, thus avoiding an external incision, may become an appealing alternative to this approach. As our group has shown, nontraumatic endoscopic inspection of the seminal ducts is feasible in the human cadaver. We investigated the feasibility and reliability of occlusion using several commercially available medical sealing agents in the porcine vas deferens (VD). Methods and Main Outcome Measures: Tests were conducted using 25 porcine spermatic ducts (10 cm length) ex vivo. The explanted specimens were fixed and cannulated using the Seldinger technique. We administered 5 different agents (n-butyle-2-cyanoacrylate, n-butyle-2-cyanoacrylate in combination with a platinum vascular coil, Tissucol Duo S®, Gelita Spon® and AFP Plug®) endoluminally. Tightness was evaluated after 5, 15, 60, 360, 720, and 1,440 min for each of the five grades, respectively, using a solution of methylene blue and saline injected under controlled pressure of 300 mm Hg followed by histological examination. Results: All agents were administered into the porcine seminal ducts (4 out of 5 via a ureteric catheter). Gelita Spon® and Tissucol Duo S® did not occlude the lumen sufficiently, whereas n-butyle-2-cyanoacrylate, n-butyle-2-cyanoacrylate in combination with coil, Tissucol Duo and AFP Plug® performed satisfactorily. In particular, cyanoacrylate combined with a coil was able to close the seminal duct tightly and for a long time. Histological findings confirmed this sealant's gapless adhesion. AFP Plug® application revealed similarly good results. However, its form needs to be optimized to ensure its suitability for endoscopic use. Conclusion: Various developments regarding minimally invasive fertility control methods have been underway for decades. Further miniaturization of endoscopy and novel materials may pave the way for endoscopic fertility control in males in the future. We demonstrated the potential of commercially available medical sealing agents to reliably occlude the porcine VD.

Vasectomy Affects Cysteine‐Rich Secretory Protein Expression Along the Human Epididymis and Its Association With Ejaculated Spermatozoa Following Vasectomy Surgical Reversal

The epididymis is essential for the acquisition of sperm fertilizing ability and forward motility. After vasectomy, the flux and composition of the epididymal fluid are modified, causing possible sequelae to the occluded excurrent duct. Some of these sequelae may not be reversible following vasovasostomy, affecting sperm physiology and their fertilizing ability. We previously demonstrated that the epididymal expression in men of a major glycoprotein secreted by the epididymis, cysteine-rich secretory protein 1 (CRISP1), and its encoding mRNA are affected by vasectomy. In this study we showed that following vasectomy, the increased level of CRISP1 is not due to a secretory defect but to its accumulation in the intraluminal compartment of the cauda epididymidis. Western blot analyses were performed to determine the amount of CRISP1 associated with spermatozoa of men who had undergone surgical vasectomy reversal. Spermatozoa of vasovasostomized men are characterized by a significant increase (P < .05) in CRISP1 levels when compared with normal donors. There was no linear correlation between CRISP1 levels and the period of time elapsed between vasectomy and vasovasostomy. CRISP1 was also present in seminal plasma of normal and vasovasostomized men, but not in vasectomized individuals. The soluble concentration of CRISP1 was significantly higher (P < .05) in seminal plasma of vasovasostomized men when compared with normal men. Knowing that one of the proposed functions of CRISP1 is to modulate sperm capacitation, we evaluated the level of tyrosine protein phosphorylation of 2 AXAP proteins of the fibrous sheath, p81 and p105. Spermatozoa of vasovasostomized men were characterized by a 50% increase of protein tyrosine phosphorylation when compared to spermatozoa of normal men (P < .05). Our results are discussed with regard to the fertilizing ability of ejaculated spermatozoa of some vasovasostomized men.

Genetic vasectomy—Overexpression of Prm1‐EGFP fusion protein in elongating spermatids causes dominant male sterility in mice

Transgenic mice are vital tools in both basic and applied research. Unfortunately, the transgenesis process as well as many other assisted reproductive techniques involving embryo transfer rely on vasectomized males to induce pseudopregnancy in surrogate mothers. Vasectomy is a surgical procedure associated with moderate pain and must be carried out under full anaesthesia by qualified personnel. Eliminating the need for vasectomy would be beneficial from the economic and animal welfare point of view. Our aim was to develop a transgene-based alternative to the surgical vasectomy procedure. We generated several transgenic mouse lines expressing a Protamine-1 (Prm1) EGFP fusion protein under the transcriptional and translational regulatory control of Prm1. Male mice from lines showing moderate transgene expression were fully fertile whereas strong overexpression of the Prm1-EGFP fusion protein resulted in complete and dominant male sterility without affecting the ability to mate and to produce copulatory plugs. Sterility was due to impaired spermatid maturation affecting sperm viability and motility. Furthermore, sperm having high Prm1-EGFP levels failed to support preimplantation embryonic development following Intracytoplasmic Sperm Injection (ICSI). The "genetic vasectomy system" was further improved by genetically linking the dominant male sterility to ubiquitous EGFP expression in the soma as an easy phenotypic marker enabling rapid genotyping of transgenic males and females. This double transgenic approach represents a reliable and cost-effective "genetic vasectomy" procedure making the conventional surgical vasectomy methodology obsolete.

Protein composition of human epididymosomes collected during surgical vasectomy reversal: a proteomic and genomic approach

The epididymal epithelium secretes membranous vesicles, called epididymosomes, with which a complex mixture of proteins is associated. These vesicles transfer to spermatozoa selected proteins involved in sperm maturation. Epididymosomes in the human excurrent duct have been described, but their protein composition and possible functions are unknown. Epididymosomes were collected during vasovasostomy procedures, purified and submitted to liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry. From all the mass spectra generated, 1022 peptides allowed the identification of 146 different proteins. Identification of some of these proteins was confirmed by western blots. Furthermore, western blot showed that the protein composition of epididymosomes differed from that characterizing prostasomes; membranous vesicles secreted by the prostate. Organization of the epididymosomes proteome according to common functional features suggests that epididymosomes have multiple functions. In order to understand the origin of epididymosomes collected distally, microarray databases of caput, corpus and cauda epididymidis were analysed to determine where along the excurrent duct the encoded proteins associated to epididymosomes are synthesised. Results suggest that some proteins synthesized in the caput and corpus epididymidis are associated with epididymosomes collected distally. Epididymosomes thus transit along the excurrent duct, and vesicles collected distally represent a mixed population.

Effects of Vasectomy on Gene Expression Profiling along the Human Epididymis1

Worldwide, almost 100 million men rely on vasectomy for male contraceptive purposes. Due to changes in their personal lives, an increasing number of these men request surgical vasectomy reversal. Unfortunately, a significant proportion of these men remain infertile, despite the reestablishment of patent ducts, possibly due to epididymal damage caused by vasectomy. In animal models, vasectomy affects different epididymal physiological and biochemical parameters. However, the consequences of vasectomy on epididymal function are poorly understood. Furthermore, results obtained with animal models cannot be extrapolated to humans to understand the consequences of vasectomy on epididymal function. Gene expression along the epididymis is highly regulated. We previously showed that the human epididymal expression pattern of two genes is altered after vasectomy. To complete the list of epididymal genes affected by vasectomy, we analyzed the epididymal gene expression pattern of three vasectomized donors using the Affymetrix human GeneChip U133 Plus 2. These results were compared with the gene expression pattern of three "normal" donors. The data generated allowed the identification of many human epididymal genes for which expression is modified after vasectomy. Quantitative (Qt)-PCR and Western blot analysis of six selected genes known to be expressed in specific epididymal segments were performed. The Qt-PCR results confirmed the selected transcripts expression pattern deduced from microarray data. However, Western blot analysis revealed some differences in protein distribution along the epididymis when compared with the encoding transcripts expression pattern. These results contribute to an understanding of the reasons why fertility is not recovered in vasovasostomized men, even though spermogram values suggest surgical success of vasectomy reversal.

Epididymal obstruction results in isolated sperm heads in post‐vasectomy rats

The aim of this study was to determine if, following vasectomy, epididymal obstruction resulted in changes in vasal stump fluid using a rat vasectomy model. One hundred and twenty-two mature male rats underwent bilateral surgical vasectomy and subsequent unilateral epididymal obstruction. Animals were randomly assigned to one of the five cohorts, which determined the time to kill and vasal fluid assessment. Numbers of whole sperm and sperm heads were compared between the obstructed and non-obstructed sides. Parametric analysis of microscopic vasal fluid findings was performed using a paired t-test. Whole sperm and sperm heads were detected bilaterally among the initial five cohorts. On the obstructed epididymis side, percentage of whole sperm dropped from 36.9% to less than 1% and sperm heads increased from 63.2 to 99.7% at 12 weeks post-obstruction (p < 0.05 at each time interval). On the unobstructed side, percentage of whole sperm rose from 66.3 to 89.5% and sperm heads dropped from 33.7 to 10.5% (p < 0.05 at each time interval). At 12 weeks, the difference between the obstructed and non-obstructed sides for both percentage and quantity of whole sperm and heads was significant with a p value of <0.001. In this rat model, following vasectomy and subsequent epididymal obstruction, testicular vasal stump fluid will contain progressively diminishing numbers of whole sperm and increases in the percentage and absolute numbers of sperm heads.

HE1/NPC2 status in human reproductive tract and ejaculated spermatozoa: consequence of vasectomy

We have previously demonstrated that the amount of HE1/NPC2 mRNA and protein expressed in the human epididymis is decreased under vasectomy. In this study, western blot analyses showed that many vasovasostomized men are characterized by high HE1/NPC2 levels in spermatozoa when compared with fertile donors. HE1/NPC2 association with sperm from vasovasostomized men was not related to low motility per se as spermatozoa from asthenospermic men have HE1/NPC2 levels similar to those in normal fertile semen samples. Spermatozoa from vasovasostomized men with high amount of HE1/NPC2 are characterized by higher concentration of cholesterol and more lipid raft domains. HE1/NPC2 is secreted in different glycoforms by different tissues of human male reproductive tract. These forms are due to variation in N-glycosylation, and only the deglycosylated form is associated with spermatozoa from some vasovasostomized men. Compared with normal men, seminal plasma of vasectomized men is characterized by a major decrease in immunodetectable HE1/NPC2 without change in the glycosylation pattern. Following surgical vasectomy reversal, seminal plasma HE1/NPC2 was found in similar amounts to the ones characterizing normal men. Considering the potential role of HE1/NPC2 in cholesterol transport during sperm maturation, unusual high levels of this protein associated with spermatozoa of vasovasostomized men may reflect epididymal sequelae occurring when the vas deferens is obstructed.

Polyol Pathway in Human Epididymis and Semen

Two enzymes are involved in the polyol pathway: an aldose reductase that reduces glucose in sorbitol followed by its oxidation in fructose by sorbitol dehydrogenase. It has been previously shown that both enzymes are presented in the bovine epididymis, where they are associated with membranous vesicles called epididymosomes. Based on the distribution of these enzymes, it has been hypothesized that the polyol pathway can modulate sperm motility during the epididymal transit. In the present study, polyol pathway was investigated in semen and along the epididymis in humans in order to determine if sperm maturation can be associated with this sugar pathway. Western blot analysis shows that both aldose reductase and sorbitol dehydrogenase are associated with ejaculated spermatozoa and prostasomes in humans. These enzymes are also associated with epididymosomes collected during surgical vasectomy reversal. Western blot, Northern blot, and reverse transcription-polymerase chain reaction analysis show that aldose reductase and sorbitol dehydrogenase are expressed at the transcriptional and translational levels along the human epididymis. Unlike what occurs in the bovine model, distribution of these enzymes is rather uniform along the human excurrent duct. Immunohistological studies together with Western blot analysis performed on epididymosomes preparations indicate that the polyol pathway enzymes are secreted by the epididymal epithelium. These results indicate that the polyol pathway plays a role in human sperm physiology.

Vasectomy surgical techniques in South and South East Asia

BackgroundSimple ligation of the vas with suture material and excision of a small vas segment is believed to be the most common vasectomy occlusion technique performed in low-resource settings. Ligation and excision (LE) is associated with a risk of occlusion and contraceptive failure which can be reduced by performing fascial interposition (FI) along with LE. Combining FI with intra luminal thermal cautery could be even more effective. The objective of this study was to determine the surgical vasectomy techniques currently used in five Asian countries and to evaluate the facilitating and limiting factors to introduction and assessment of FI and thermal cautery in these countries.MethodsBetween December 2003 and February 2004, 3 to 6 major vasectomy centers from Cambodia, Thailand, India, Nepal, and Bangladesh were visited and interviews with 5 to 11 key informants in each country were conducted. Vasectomy techniques performed in each center were observed. Vasectomy techniques using hand-held, battery-driven cautery devices and FI were demonstrated and performed under supervision by local providers. Information about interest and open-mindedness regarding the use of thermal cautery and/or FI was gathered.ResultsThe use of vasectomy was marginal in Thailand and Cambodia. In India, Nepal, and Bangladesh, vasectomy was supported by national reproductive health programs. Most vasectomies were performed using the No-Scalpel Vasectomy (NSV) technique and simple LE. The addition of FI to LE, although largely known, was seldom performed. The main reasons reported were: 1) insufficient surgical skills, 2) time needed to perform the technique, and 3) technique not being mandatory according to country standards. Thermal cautery devices for vasectomy were not available in any selected countries. Pilot hands-on assessment showed that the technique could be safely and effectively performed by Asian providers. However, in addition to provision of supplies, introducing cautery with FI could be associated with the same barriers encountered when introducing FI in combination with LE.ConclusionFurther studies assessing the effectiveness, safety, and feasibility of implementation are needed before thermal cautery combined with FI is introduced in Asia on a large scale. Until thermal cautery is introduced in a country, vasectomy providers should practice LE with FI to maximize effectiveness of vasectomy procedure.

Noninvasive vasectomy using a focused ultrasound clip: thermal measurements and simulations.

Conventional surgical vasectomy may lead to complications including bleeding, infection, and scrotal pain. Noninvasive transcutaneous delivery of therapeutic focused ultrasound has previously been shown to thermally occlude the vas deferens. However, skin burns and inconsistent vas occlusion have presented complications. This study uses bio-heat transfer simulations and thermocouple measurements to determine the optimal ablation dosimetry for vas occlusion without skin burns. A 2-rad ultrasound transducer mounted on a vasectomy-clip-delivered ultrasound energy at 4 MHz to the canine vas deferens co-located at the focus between the clip jaws. Chilled degassed water was circulated through an attached latex balloon, providing efficient ultrasound coupling into the tissue and active skin cooling to prevent skin burns. Thermocouples placed at the vas, intradermal, and skin surface locations recorded temperatures during ablation. Procedures were performed with transducer acoustic powers of 3-7 W and sonication times of 60-120 s on both the left and right vas deferens (n = 2) in a total of four dogs (precooling control, 3 W/120 s, 5 W/90 s, 7 W/60 s). Measurements were compared with bio-heat transfer simulations modeling the effects of variations in power and sonication time on tissue temperatures and coagulation zones. Active skin cooling produces a thermal gradient in the tissue during ablation, allowing sufficient thermal doses to be delivered to the vas without skin burns. However, low-power, long-duration heating produced excessive tissue necrosis due to thermal diffusion, while high power and short heating times reduced the therapeutic window and produced skin burns presumably due to direct ultrasound absorption. Both simulations and experiments suggest that a therapeutic window exists in which thermal occlusion of the vas may be achieved without the formation of skin burns in the canine model (power = 5-7 W, surface intensity = 1.4-1.9 W/cm2, time = 20-50 s). This range of ablation parameters will help guide future experiments to refine incisionless vasectomy using focused ultrasound.

Gossypol blood levels and inhibition of spermatogenesis in men taking gossypol as a contraceptive: A multicenter, international, dose-finding study

The safety and efficacy of gossypol continues to be controversial. The aim of this study was to evaluate gossypol as a contraceptive pill for men at doses lower than those previously prescribed and in men from various ethnic origin. A total of 151 men from Brazil, Nigeria, Kenya, and China were divided into two groups. Both groups received 15 mg gossypol/day for 12 or 16 weeks to reach spermatogenesis suppression. Subjects were then randomized to either 7.5 or 10 mg/day for 40 weeks. In addition, 51 men were enrolled as a control group. In all, 81 subjects attained spermatogenesis suppression. Only one man discontinued treatment because of tiredness. Potassium levels fluctuated within the normal range. FSH increased consistently. Testicular volume decreased, but after discontinuation, values returned to levels not statistically different from admission. Of 19 subjects on the 7.5 mg/day dose group, 12 recovered sperm counts >20 million/mL within 12 months of discontinuing gossypol. In the 10 mg/day group, sperm counts recovered in only 10 of 24 subjects. Eight of the 43 patients remained azoospermic 1 year after stopping gossypol. All men diagnosed with varicocele failed to reverse spermatogenesis suppression. Gossypol blood levels indicated that sperm suppression occurs independently of concentration, whereas spermatogenesis recovery appears to be concentration-dependent. Gossypol may become a medical alternative to surgical vasectomy when the delay in onset of infertility is acceptable. When taken for 1 year, gossypol causes no reduction in sexual desire or frequency of intercourse. The possibility of reversal, occurring in 51% of the men on this regimen within 1 year after stopping gossypol, is an advantage of this compound as compared with surgical sterilization in many parts of the world.

Introduction of non-scalpel vasectomy at the Mexican Social Security Institute

Since government approval of the Mexican Family Planning Program in 1977, the Mexican Social Security Institute (IMSS) has been one of the major family planning service providers in Mexico. No-scalpel vasectomy, a refined surgical vasectomy technique developed and widely used in China, has been introduced in 20 countries, including Mexico. This paper will describe the experience of the IMSS in introducing the no-scalpel vasectomy technique into its service delivery system, and will review the clinical and programmatic experience, and the impact of no-scalpel vasectomy on client and provider attitudes toward vasectomy.

Transcutaneous procedures for male sterilization.

Outside the People's Republic of China transcutaneous methods of male sterilization are not widely used, even though there are advantages of these methods over surgical vasectomy procedures. Methods of transcutaneous sterilization (intratesticular, intraepididymal, and intravasal) that have been evaluated in men are reviewed.

Frozen semen — A poor form of fertility insurance

Two previously fertile males have requested surgical vasectomy reversal after divorce and failure of their new younger wives to conceive by repeated inseminations with their stored frozen semen. The sperm had been preserved for six and five years, respectively, as “fertility insurance” prior to sterilization by bilateral vasectomy. It is clear that the storage of fertile semen does not guarantee future fertility.

A factor analysis of contraceptive preferences.

Summary Factor analysis of acceptability ratings given to 10 contraceptive methods by 178 Ss (78 male, 100 female) identified four groupings: (a) coitusdependent—condom, diaphragm, and foam or jelly; (b) surgical—vasectomy and tubal ligation; (c) coitus-inhibiting—abstinence, rhythm, and withdrawal; and (d) coitus-independent—intrauterine device and pill. Factor scores were computed and correlated with variables from the Adjective Check List, California F Scale, California Psychological Inventory, College Vocabulary Test, Miller-Fisk Sexual Knowledge Questionnaire, and Rotter's locus-of-control scale. Although the yield of significant correlations was modest, several heuristic patterns of covariation with the personality and cognitive measures were noted.