Medical Professionals

Full Publications

Effect of Vaginal Lubricants on Sperm Motility and Chromatin Integrity: A Prospective Comparative Study - Fertility & Sterility. 2008 Feb;89:375-379

Agarwal A, Deepinder F, Cocuzza M, Short RA, Evenson DP. Reproductive Research Center, Glickman Urological Institute and Department of Obstetrics-Gynecology, Cleveland Clinic, Cleveland, Ohio

Objective: To evaluate the effect of vaginal lubricants Pre~Seed®, FemGlide®, Astroglide®, and Replens® on human sperm motility and chromatin integrity. Design: Prospective, comparative, in vitro study. Setting: Andrology laboratory at tertiary care hospital. Patient(s): Thirteen normozoospermic donors. Intervention(s): Semen samples from 13 subjects were incubated in human tubal fluid media (HTF) controls and 10% (vol/vol) of Pre~Seed®, FemGlide®, Astroglide®, and Replens® lubricants. After 30 minutes, progressive sperm motility was assessed by light microscopy. Semen samples of 12 patients were placed in positive control (HTF), negative control (10% K-Y Jelly® lubricant), and 10% vol/vol Pre~Seed® and FemGlide® lubricants. After 4 hours culture, spermatozoa were analyzed for percent DNA fragmentation index with use of the acridine orange-based sperm chromatin structure assay. Main Outcome Measure(s): Sperm motility and percent DNA fragmentation index. Results: Percent motility did not differ significantly between HTF controls and Pre~Seed®, whereas FemGlide®, Replens®, and Astroglide® lubricants demonstrated a significant decrease in motility. There was no significant difference in percent DNA fragmentation index between the HTF controls and Pre~Seed®, but a significant decline in sperm chromatin quality occurred with FemGlide® and K-Y Jelly®. Conclusion: Pre~Seed® does not cause a significant decrease in progressive sperm motility or chromatin integrity in contrast to other lubricants used by couples.

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Mucosal Irritation Potential of Personal Lubricants Relates to Product Osmolality as Detected by the Slug Mucosal Irritation Assay - Sexually Transmitted Diseases. 2008 May;35:512-516

Els Adriaens, PhD; Jean Paul Remon, PharmD. Laboratory of Pharmaceutical Technology, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium

BACKGROUND: The slug mucosal irritation assay has recently been used as a sensitive measure of mucus membrane tolerance for vaginal microbicide products and carriers. In the current study, it was determined whether mucosal irritation potency of personal lubricants is related to varying product osmolalities.
METHODS: Five commercial lubricants with an osmolality range were evaluated using the previously validated slug mucosal irritation assay. Specifically, Arion lusitanicus were treated with lubricants over 5 days to quantify mucus production and tissue damage, allowing assignment of each product into an irritation potency category (none, mild, moderate, or severe). RESULTS: The irritation potency (assessed by the mucus production) of the lubricants showed a significant, quadratic relationship with the product osmolality (P = 0.001; R (2) = 0.99). Femglide, a hypo-osmotic lubricant (32 mOsm/kg), caused a negative mucus production. Pré, an iso-osmotic lubricant (316 mOsm/kg), caused no changes. Two moderately hyperosmotic lubricants, Replens and K-Y jelly (2143 and 2463 mOsm/kg), induced mild and moderate irritation, respectively. The highly hyperosmotic lubricant Astroglide (5848 mOsm/kg) resulted in severe irritation and tissue damage. CONCLUSIONS: Commonly used personal lubricants show a full range of mucosal irritation potential, which is related to product osmolality.

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Sperm Toxicity of ‘Nonspermicidal’ Lubricant and Ultrasound Gels Used in Reproductive Medicine - In preparation for submission 2010

Josefina Vargas, M.Sc.; Michel Crausaz, M.Sc.; Alfred Senn, Ph.D.; Marc Germond, M.D. Fondation F.A.B.E.R., Rue de la Vigie 5, 1003 Lausanne, Switzerland

Objective: To compare sperm toxicity of four commercial “non-spermicidal” gels used in Reproductive Medicine including: Aquasonic Ultrasound Gel, Felis Lubricant, Pre-Seed Lubricant and Replens Moisturizer, in a range of concentrations (0.083% - 8.3%) believed to be physiologically relevant. Results: Sperm toxicity was observed as shown by significant declines in motility over the 24-hr assay following incubation with Aquasonic, Felis and Replens at higher gel concentrations. Although Replens, even at only 0.83% v/v resulted in sperm toxicity. Pre-Seed alone was not toxic to sperm at any concentration, including the highest (8.3%). Conclusions: Three of the “nonspermicidal” gels were toxic to sperm, including Aquasonic Gel which is widely used for transvaginal ultrasound during ovulation. Pre-Seed alone did not cause sperm toxicity at any time or concentration. It is appropriate for use by patients trying to conceive or clinicians during fertility procedures, including facilitating vaginal insertion of ultrasound probes.

Medical Review Articles

Optimizing Natural Fertility - Fertility & Sterility. 2008 Nov;90(Suppl 3):S1-S6

Practice Committee of the American Society for Reproductive Medicine in collaboration with the Society for Reproductive Endocrinolgy and Infertility

Synopsis: Some vagin al lubricants may decrease fertility, based on their observed effects on sperm survival in vitro. Whereas commercially available water-based lubricants (e.g., Astroglide®, KY Jelly®, and Touch®) inhibit sperm motility in vitro by 60% to 100% within 60 minutes of incubation, canola oil has no similar detrimental effect (32). KY Jelly, olive oil, and saliva diluted to concentrations even as low as 6.25% adversely affect sperm motility and velocity.... Hydroxyethylcellulose-based lubricants such as Pre-Seed® (INGfertility, Valleyford, WA) also have no demonstrable adverse impact on semen parameters (35). Although there is no evidence to indicate that use of any vaginal lubricant decreases fertility, it seems prudent to recommend... hydroxyethylcellulose-based lubricants when they are needed.

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Dyspareunia and Vaginal Dryness after Breast Cancer Treatment - SRM Sexuality, Reproduction & Menopause. 2008 Aug;6:18-22

Doreen Leyden Wiggins, MD1 & Don S. Dizon, MD2. 1Clinical Assistant Professor, Program in Women's Oncology and 2Assistant Professor, Obstetrics and Gynecology and Medicine. Co-Directors, Center for Sexuality, Intimacy & Fertility, Women and Infants Hospital of Rhode Island, Providence, RI

An animal model using slugs has been validated to test mucosal irritation in human mucosal membranes. A recent study using the slug mucosal irritation assay determined that product osmolality was an important component to mucosal tolerability. The iso-osmotic lubricant (Pre-Seed) caused no changes and, therefore, was the best tolerated. The hypoosmotic lubricant (Femglide) caused negative mllCllS production, thereby decreasing natural response. Two moderately hyperosmotic lubricants (Replens, K-Y jelly) induced mild and moderate irritation, respectively. The highly hyperosmotic lubricant (Astroglide) resulted in severe irritation and tissue damage.Pre-Seed has been marketed to couples who need lubrication that does not inhibit fertility.

Medical Meeting Presentations

Safety and Anti-HIV Activity of Over-the-Counter Lubricant Gels - International Microbicide Conference, Pittsburgh, PA, 2010

Julie Russo, Lisa C. Rohan, Bernard Moncla, Ratiya Pamela Na Ayudhya, Lin Wang, Marilyn Cost, Kara Pryke, Marc-André LeBlanc, Jim Pickett, and Charlene S. Dezzutti Magee-Women’s Research Institute, University of Pittsburgh, Pittsburgh, PA; University of Pittsburgh, Pittsburgh, PA; International Rectal Microbicide Advocates, Chicago, IL

Background: Because lubricants may decrease trauma during coitus, it is hypothesized that they could aid in the prevention of HIV acquisition. However, the safety and anti-HIV activity is currently unknown for over-the counter (OTC) lubricant gels. Methods: Based on an International Rectal Microbicide Advocates survey, 6 OTC lubricant gels were tested: 5 aqueous-based (Astroglide, Elbow Grease, ID Glide, KY Jelly, and PRÉ) and 1 condom compatible silicone based (Wet Platinum). Formulation characteristics (pH, osmolarity, and viscosity) were determined. Viability of Lactobacillus species and cells [Caco-2 (colorectal), HEC-1-A (uterine), and TZM-bl (cervical) epithelial cell lines and peripheral blood mononuclear cells (PBMCs)] exposed to the lubricants was evaluated. For cells, dilutions of each aqueous-based lubricant were made; Wet Platinum was used undilute. Transepithelial resistance of Caco-2 and HEC-1-A cell lines was measured to determine the impact of lubricants on epithelial cell monolayers. The anti-HIV activity was tested with the TZM-bl cell line. Colorectal and ectocervical safety was evaluated by the MTT assay and histology after apical application to polarized explant cultures. Results: PRÉ was pH 7, isosmolar, with moderate viscosity. Elbow Grease, ID Glide, and KY Jelly were pH 4 to 5, 9 to 13-fold above isosmolar, with varying degrees of viscosity. Astroglide was pH 4, 21-fold above isosmolar, with low viscosity. KY Jelly which contains chlorhexidine had a complete loss of Lactobacillus viability, but the other lubricants had < log10 loss of bacteria and were considered safe. PRÉ was not toxic up to 1:10 dilution for the PBMCs and cell lines. Elbow Grease, ID Glide, and KY Jelly were not toxic up to 1:100 to 1:200 dilutions. Astroglide was not toxic up to 1:1500 dilution. Wet Platinum had no toxicity. PRÉ had no impact on the epithelial cell monolayers whereas the other aqueous-based lubricants disrupted the epithelial cell monolayers. All lubricants retained colorectal and ectocervical explant viability by MTT assay. Histology showed intact epithelium for PRÉ and Wet Platinum, while epithelial striping was observed for Astroglide, Elbow Grease, ID Glide, and KY Jelly. Lubricants had no measurable anti-HIV activity. Conclusions: Our data suggests that PRÉ and Wet Platinum were safest. The hyperosmolar nature of the other lubricant gels was associated with cellular toxicity and may lead to increased risk of HIV infection.

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Effect of Lubricants Developed for Fertility Markets on In Vitro Fertilization and Embryo Development - American Society of Andrology, Philadelphia PA, 2009

Raymond W. Wright Jr, PhD. Washington State University, Center for Repro Biology, Pullman, WA

Introduction: Traditional lubricants damage sperm and should not be used when pregnancy is desired. Newer products have been developed for this consumer/patient group. Bovine and human embryos share similar paternal sperm regulatory pathways, making this species a model for detection of sublethal sperm damage. Objective: Experiments were done to evaluate in vitro fertilization and embryo development following bull sperm exposure to lubricants developed for the fertility market. Methods: Cryopreserved bull sperm was washed, resuspended in medium and placed into one of 4 treatments. These were: 1) Control medium; 2) Pré® Lubricant (Pré); 3) ConceiveEase™ (CE) or 4) PreConceive plus™ (PC). Lubricants were mixed with sperm at 10% v/v & incubated for 30 min at body temperature. Then sperm from each treatment were placed into fertilization wells with mature bovine oocytes. At 8 hr, putative zygotes were transferred into development medium and further incubated. At 32 hr of culture, dividing embryos were counted (% fertilized oocytes). Embryo development (%) was determined by the number of morulae and blastocysts on Day 7. Mean percent fertilization and embryo development rates resulting from sperm in each treatment were compared to fertilization and embryo development percentages resulting from sperm in control media (no lubricant) using the Friedman’s. Results: CE and PC exposure resulted in sperm that a) were significantly less able to participate in fertilization and b) had reduced embryo development rates compared to that seen for sperm in control media. Sperm exposed to Pré did not differ in these outcomes from sperm in controls.
Conclusion: Sperm contact with Pré did not interfere with fertilization or embryo development, whereas other lubricants caused significant declines in these end points. Several lubricants marketed to trying- to-conceive couples, caused a large decline in the subsequent ability of embryos to develop normally after a 30 min pre-fertilization exposure of sperm to the lubricants. The reasons for these differences require additional study.
Funded in part by INGfertility. Pré Lubricant - INGfertility, Valleyford, WA (Also, sold as Pre~Seed® Intimate Moisturizer & Lubricant); FertilityCare™ - Marco D’Polo, Ingleburn, NSW, AU; ConceiveEase™ - Sepal Reproductive Devices, Boston, MA; Pre Conceive plus™ - Lake Consumer Products, Jackson WI

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Animal Model Study of a New Patient Lubricant’s Affect on In Vitro Fertilization & Embryo Development - American Society of Andrology, Orlando FL, April 2007

RW Wright Jr, PhD. Center for Reproductive Biology, Washington State University, Pullman, WA

Experiments were done to evaluate in vitro fertilization and embryo development following sperm exposure to products used to lubricate devices in fertility medicine including: KY® Gel, Aquasonic® Ultrasound Gel and Pre’® (a new Patient Lubricant recently cleared for use during fertility interventions). Bovine in vitro fertilization and embryo culture methods are standard and have been proposed as an excellent model for gamete toxicity studies (ReprodBioMed Online 2002;4:170-5). In this study, cryopreserved bull sperm (from a single bull) were routinely washed, resuspended in a TALP medium and placed into one of 5 treatments. These included: 1) Control sperm in medium alone; or sperm medium suspensions with the following added (v/v) 2) 10% Pre’® lubricant; 3) 50% Pre’® lubricant; 4) 10% KY®; or 5) 10% Aquasonic® Gel. Sperm were incubated in treatments for 30 min at body temperature, and placed into fertilization wells with mature oocytes (1 x 106 sperm cells per well). At 8 hrs, putative zygotes were transferred into embryo culture medium and further incubated.  At 32 hr of culture, dividing embryos were counted (% fertilization in each treatment). Final development rates were evaluated on Day 7 (post IVF) to determine the % of total oocytes that had developed to the morula or blastocyst stage. ANOVA was used to compare the % fertilization of oocytes & the % of normal embryo development resulting from sperm in each treatment (as seen in Table below, data are mean +/- sd).

In Vitro Fertilization & Embryo Development After Sperm Exposure

Treatment	Total Oocyte Number	% Fertilized Oocytes (+ sd)	% Embryos Developing (+ sd)
Control Medium	80	61(5)a	40(9)a
Pre’® 10%	80	60(8)a	39(8)a
Pre’® 50%	80	59(6)a	43(10)a
KY 10%	80	23(6)b	6(5)b
Aquasonic10%	80	0c	0c

a,b,c denote means that differ within column by p<0.0001(ANOVA).

Pre’® Patient Lubricant did not interfere with the ability of sperm to fertilize oocytes or support embryo development in vitro (using a bovine model) even at high concentrations. Conversely, KY® and Aquasonic® significantly impacted the ability of sperm to fertilize oocytes, and allow normal embryo development.

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Changes in Sperm Motility and Chromatin Integrity Following Contact with Vaginal Lubricants - American Society for Reproductive Medicine, Montreal Quebec, October 2005

Agarwal A, Deepinder F, Cocuzza M, Short RA, Evenson DP. Reproductive Research Center, Glickman Urological Institute and Department of Obstetrics-Gynecology, Cleveland Clinic, Cleveland, Ohio

OBJECTIVE: To evaluate the effect of vaginal lubricants Pre-Seed®, FemGlide®, Astroglide®, and Replens® on human sperm motility and chromatin integrity. DESIGN: Prospective, comparative, in vitro study. SETTING: Andrology laboratory at tertiary care hospital. PATIENT(S): Thirteen normozoospermic donors. INTERVENTION(S): Semen samples from 13 subjects were incubated in human tubal fluid media (HTF) controls and 10% (vol/vol) of Pre-Seed®, FemGlide®, Astroglide®, and Replens® lubricants. After 30 minutes, progressive sperm motility was assessed by light microscopy. Semen samples of 12 patients were placed in positive control (HTF), negative control (10% K-Y Jelly® lubricant), and 10% vol/vol Pre-Seed® and FemGlide® lubricants. After 4 hours culture, spermatozoa were analyzed for percent DNA fragmentation index with use of the acridine orange-based sperm chromatin structure assay. MAIN OUTCOME MEASURE(S): Sperm motility and percent DNA fragmentation index. RESULTS: Percent motility did not differ significantly between HTF controls and Pre-Seed®, whereas FemGlide®, Replens®, and Astroglide® lubricants demonstrated a significant decrease in motility. There was no significant difference in percent DNA fragmentation index between the HTF controls and Pre-Seed®, but a significant decline in sperm chromatin quality occurred with FemGlide® and K-Y Jelly®. CONCLUSION: Pre-Seed® does not cause a significant decrease in progressive sperm motility or chromatin integrity in contrast to other lubricants used by couples.

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The Effects of Vaginal Lubricants and Moisturizers on Computer Assisted Sperm Analysis (CASA) Parameters Associated with Cervical Mucus Penetration - American Society for Reproductive Medicine, Philadelphia PA, October 2004

JE Ellington and J Schimmels. INGfertility, Spokane, WA & Washington State University, Spokane, WA

Objective: The incidence of vaginal dryness is increased in trying-to-conceive (TTC) couples; however, numerous papers have cited the detrimental effect of common vaginal lubricants and moisturizers on sperm motility. To date, studies have not been done using CASA to evaluate the effects of lubricant products on the motion characteristics of sperm thought to be associated with cervical mucus transport. Specifically, samples of sperm with mean average path velocity (VAP), % straightness (STR) and Amplitude of Lateral Head Displacement (ALH) exceeding a predetermined level have recently been proposed to have a superior likelihood of good cervical mucus penetration in vivo. Numerous studies have found a correlation between such ability of sperm to penetrate cervical mucus and pregnancy outcomes. The current study evaluated sperm motion parameters following contact with several vaginal lubricants/moisturizers, to determine their impact on CASA outcomes associated with good cervical mucus penetration, as well as overall motility. Design: Prospective, comparative, in-vitro study.

Materials and Methods: Raw semen from 25 normospermic donors was diluted 1:1 with Human Tubal Fluid. Each sample was then divided into one ml aliquots and placed into multiple culture wells. Vaginal lubricants/moisturizers as shown in the Table below were applied across these wells to achieve a final 10% v/v concentration, and incubated at 37oC for 30 min. CASA of sperm from these treatments and those in a control well (with no lubricant) was then performed. Samples in each treatment fulfilling all of the following parameters were given a positive penetration score (e.g. likely to penetrate cervical mucus well): VAP > 25 micron/sec; STR >80%; and ALH >2.5 microns. Positive penetration scores were reported as a percent of all samples tested and compared between the treatments. Additionally, mean outcomes in each treatment were determined and compared for the individual CASA parameters, as well as for the overall percentage of motile sperm. Results: The mean motion characteristics for these specific CASA criteria (+/- SEM) and percent samples with a positive penetration score are shown in the Table below. Means with differing superscripts differ from the control at p<0.05.

Replens caused the media to abruptly turn very acidic and opaque. Further, sperm clumping occurred to the point that meaningful CASA data could not be generated. Conclusion: Within 30 minutes of exposure, a 10% v/v concentration of the most commonly used lubricant products significantly decreased sperm motility and penetration scores. The percentage of samples with a positive penetration score was significantly improved with Pre~Seed® as compared to the other treatments. Products used to alleviate vaginal dryness that negatively affect sperm motility and transport should be avoided by TTC couples. Studies to determine the in vivo impact of vaginal lubricants/moisturizers on cervical mucus penetration are ongoing. Support: NICHD SBIR to INGfertility

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Prevalence of Vaginal Dryness in Trying-to-Conceive Couples - Pacific Coast Reproductive Society, Rancho Mirage CA, April 2003

JE Ellington and RA Short. INGfertility, Spokane, WA & Washington State University, Spokane, WA

Dyspareunia, primarily due to vaginal dryness, has been reported to occur “sometimes” or “more often” in at least 46% of all reproductive age women. However, it is currently not known if vaginal dryness is increased in trying-to-conceive (TTC) couples. Additionally, it is not known how TTC couples are managing symptoms of vaginal dryness, given numerous reports on the sperm-toxic nature of most personal lubricants and even saliva. This study was done to determine the prevalence of vaginal dryness among TTC couples, and their level of understanding of appropriate interventions for such dryness. An opt-in internet survey of 900 TTC couples was conducted over 5 months. Thirty questions regarding fertility and vaginal dryness were asked of each participant. Summary statistics for the group were compiled and analyzed. Average TTC time for the group was 7 months, with 33% TTC 1 year or more. Medical care for their fertility issues included: 23% no doctor, 13% PCP, 43% ObGyn, 16% Fertility Specialist, 4% Urologist. Most couples (78%) had no definitive diagnosis for cause of fertility problems. Most (69%) routinely used some ovulation prediction method. Only 16% were currently taking “fertility medications”. While TTC, vaginal dryness negatively affected sexual intimacy for most couples: 11% always, 35% often, 42% sometimes, 9% rarely, 3% never. Vaginal dryness episodes also increased while TTC: 19% a lot, 57% some, 23% not at all. Although 30% knew not to use a lubricant while TTC, another 26% often or always used such products. Use by this later group included mostly that of KY® (40%) and Astroglide® (19%). Only 20% of couples had ever discussed their dryness problem with a doctor. Of those that had, 75% of the doctors reiterated the sperm-toxic effects of lubricants. Rates of vaginal dryness in TTC couples appears to be as much as twice that seen in the general population. Patients are not discussing this problem with their care providers adequately. Fully one-quarter of TTC couples are utilizing personal lubricant products which reportedly are as toxic to sperm as are contraceptive jellies. Products designed specifically to relieve vaginal dryness without harming sperm, such as Pre~Seed®, are needed for use by TTC couples.

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Effects of Personal Lubricants on In Vitro Fertilization and Embryo Development
American Society of Andrology, Phoenix AZ, March 2003

RW Wright1, PhD; RA Short2, PhD; & JE Ellington3 DVM, PhD. 1Dept Animal Science & 2Health Research Center, Washington State University; and 3INGfertility, Spokane, WA

Use of personal lubricants is not recommended for couples that are trying to conceive based on several studies reporting their deleterious effect on sperm motility. In spite of this, 43% of all trying-to-conceive couples use personal lubricant products due to a high frequency of vaginal dryness. The current study was designed to compare in vitro fertilization and embryo development of bovine oocytes in the presence of moderate doses (10%) of several different products. In vitro matured cow oocytes were fertilized by bull sperm with: 10% KY Jelly®; 10% FemGlide® (labeled as "sperm compatible"); 10% Pre~Seed® (a new moisturizer developed to provide an optimal sperm environment); and control TALP IVF media. Lubricants were only present during the fertilization incubation of sperm and oocytes. The bovine IVF model allows for detection of sperm DNA damage which can inhibit embryo development. Embryos were cultured for 7 days and then scored for normal development for blastocyst (multi-cell) stage. Data are expressed as Mean (SEM).Treatment

	# Oocytes	% Fertilized	% Blasts
KY Jelly®	100	12 (2.0)a	2 (1.2)a
FemGlide®	200	72 (3.4)b	42 (0.7)b
PreSeed®	200	73 (4.6)b	47 (0.9)c
Control®	200	77 (3.4)b	44 (0.8)b,c

KY Jelly® in the fertilization medium had a very negative effect on fertilization and development (a,c differ by p<0.001), with only 2% of all eggs developing to the blastocyst stage. FemGlide® decreased embryo development as compared to the Pre~Seed® treated sperm (b,c differ by p=0.05). Pre~Seed® did not affect embryo development as compared to the control media in this model, in fact a trend for improved development was seen. Mouse embryo development studies with 10% volume of test product are routinely done as a toxicology screen for assisted reproduction media. A similar design, using cow embryos detected a harmful effect of KY Jelly® and FemGlide® on embryo development after sperm exposure to these products.

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Effect of New Intimate Moisturizer on Sperm Motility - American Society of Andrology, Phoenix AZ, March 2003

JE Ellington PhD; RA Short PhD; & J Schimmels. iNGfertility, Spokane, WA & Health Research Center, Washington State University, Spokane, WA

Numerous publications cite the deleterious effect of existing commercial lubricants on sperm motility. Additionally, 75% of trying-to-conceive couples have an increased incidence of vaginal dryness. This study compared motility parameters for human sperm (n=25 ejaculates) cultured for 30 min in HTF media with HSA (control), to which either 10% KY Jelly®; 10% Astroglide®; 10% FemGlide® (marketed as “sperm compatible”); or 10% Pre~Seed® (specifically developed to not harm sperm) were added.

Treatment	% Progressive Motility	VSL (µm/s)	VCL (µm/s)	VAP (µm/s)
Control	100a	53 (2)a	89 (3)a	59 (2)a
KY®	62 (6)b	37 (2)b	67 (2)b	40 (2)b
FemGlide®	92 (4)c	44 (4)c	79 (3)c	50 (4)c
PreSeed®	100 (5)a	51 (2)a	79 (2)c	56 (3)a,c
Astroglide®	<5	NA	NA	NA

a,b,c Superscripts show means (SEM) within a column that differ at p<0.05.

Due in part to viscosity change, all lubricants slowed sperm velocity as compared to control medium. However, sperm in Pre~Seed® retained motility equivalent to the control over the 30 min of culture, whereas sperm in the other lubricants had decreased motility (p<0.05). This effect was profound with Astroglide®. Placed side by side, FemGlide® and KY® created a distinct barrier whereby sperm in raw semen had difficulty penetrating into the products (photos available). In contrast, sperm moved freely between raw semen and the Pre~Seed®

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Polysaccharides Containing Arabinose and Galactose Decrease Oxidative Damage to Sperm In Vitro - American Society of Andrology, Montreal Quebec, June 2002

JE Ellington, SA Oliver, DP Evenson. Washington State University, Spokane, WA and South Dakota State University, Brookings, SD

Polysaccharides containing arabinose & galactose (PCAG), such as arabinogalactan, are abundant in plant gums. These PCAG have a membrane stabilizing effect in a variety of cell types. Studies were done to determine the effects of PCAG on bull sperm during freezing and culture. In Experiment 1, ejaculates from 4 bulls at a commercial AI stud were frozen in standard egg yolk buffer (EYB) or in egg yolk buffer with PCAG (PEYB). Six straws of sperm from each bull and treatment were thawed and: 1) held at 37°C for 10 min, then evaluated for membrane lipid peroxidation (TBARS assay) and sperm chromatin damage (Sperm Chromatin Structure Assay); or 2) cultured in routine TALP medium for 24h to determine sperm survival rates. Results: sperm frozen in EYB had more oxidative (p=0.03) and chromatin (p=0.01) damage after thawing than sperm in PEYB. Only 1 of 4 bulls had > 10% motile sperm at 24h of culture for sperm frozen in EYB; whereas 3 of 4 bulls had >10% motile sperm for sperm frozen in PEYB.

In Experiment 2, sperm from 5 bulls (4 straws each) frozen in standard EYB were thawed, washed and placed in TALP either w/or w/o PCAG for culture at 37°C. At 4h, sperm motility was determined and aliquots were removed to determine membrane lipid peroxidation. Results: More sperm were motile (p=0.04) and had lower oxidative damage (p=0.01) in TALP with added PCAG, than in TALP alone. Follow-up studies identified an active fraction of the PCAG between 20K and 100K which promoted sperm motility and membrane stability. Preliminary studies have shown decreased oxidative stress and chromatin damage for human sperm in culture with HTF including the PCAG. Conclusion: PCAG stabilize sperm during assisted reproduction techniques. Specifically, they appear to decrease oxidative stress and chromatin damage.

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Use of a Plant Polysaccharide Gradient to Wash Bull Sperm Improves Fertilization & Embryonic Development - International Embryo Transfer Society, Salt Lake City UT, Jan 1996

JE Ellington, SA Oliver, RW Wright, CS Schneider, AJ Benson. INGfertility & Washington State University, Spokane, WA

Experiment 1. A continuous gradient of 22% plant polysaccharides (arabinogalactan) in a buffered salt solution (Sperm Concept-INGfertility, Spokane, WA) was compared to a standard Percoll density gradient of 45 % and 90% using frozen-thawed bull sperm (Select Sires). Studies were designed to evaluate sperm recovery and performance in a routine IVF system as determined by fertilization rates and subsequent embryonic development. Frozen sperm from 4 bulls (2 replicates each) were washed through 4 ml of Sperm Concept (SC) or gradient Percoll (P) for 30 min at 300 x g. The pellet of sperm from P was washed again in a TALP medium prior to use. No extra wash step was used in the hemocytometer. Sperm motility was also evaluated both subjectively and objectively with an HTM Analyzer. All data are expressed as the mean SEM for SC or P treated sperm, respectively. Statistical analyses were conducted using ANOVA.

Conclusions: Recovery of sperm was significantly higher (p=0.02) after SC washing than with P (72 6% vs 53 5%). SC also tended (p= 0.08) to recover more of the motile sperm than did P (88 9% vs 73 6%). Overall percent motilities were high for both groups and did not differ (p=0.18; 95 0.3 % vs 90 3%).

Experiment 2. Pooled frozen bull sperm was also washed through either SC or P as above, treated routinely with 10 IU heparin/ml and placed with total of 1100 IVM oocytes over three replicates. Oocytes were obtained from slaughtered beef heifers. Cleavage rates were determined visually on Day 3 of culture in CZB medium. Embryo quality was recorded on Day 9 of coculture on BRL cells, followed by embryo staining (Hoechst 33258) and cell counts. Embryo evaluators were blind to the sperm washing treatment utilized prior to IVF. Conclusion: Fertilization rates for the oocytes were improved after washing sperm through SC versus P (p=0.001; 73 2% versus 53 3%). The percentage of fertile oocytes able to develop to blastocysts by Day 9 of culture was higher after washing sperm through SC versus P (p=0.009; 59 4% vs 26 5%). Overall production of blastocysts as a percentage of total oocytes introduced into the IVF system was greater after washing sperm through SC versus P (p=0.004; 43 4% vs 18 3%). Total cell counts for blastocysts formed after fertilization with either SC or P treated sperm did not differ (p=0.25; 85 2 vs 88 2).

Overall Conclusion: Washing frozen thawed bull sperm through SC appears to offer several advantages over P gradients. A pellet of highly motile sperm cells can be obtained in one centrifugation step. Sperm exposed to the arabinogalactan-containing SC wash show improved ability to fertilize oocytes and contributed to better embryonic development to the blastocyst stage.

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A Novel One Step Sperm Wash Product - Pacific Coast Fertility. Palm Springs CA, March 1995

JE Ellington, RW Wright, S Broder 1, AJ Benson, SA Oliver. INGfertility, Spokane, WA & 1California Cryobank, Los Angeles, CA

A continuous gradient sperm wash product containing arabinogalactan, Sperm Concept (sold as IsoCare One Step) was compared against a Percoll wash in 3 experiments.

Experiment 1: In Expt. 1, fresh semen from 4 men was divided and washed either in SC or 80% Percoll (P) for 30” at 300 x g. Pellets were then washed again in media alone and sperm evaluated. All data are expressed below as mean SEM for SC versus P. There was no difference in the % motility for recovered sperm (83 4% vs 83 3%), the motility of sperm cultured for 24 h (64 6% vs 75 6%) or in the numbers of sperm recovered (p=0.6). Based on animal model data showing a lack of toxicity of SC to sperm throughout the IVF process, the next 2 experiments were done on sperm removed directly from the SC pellet, without a second media wash step.

Experiment 2: In Expt. 2, SC was compared to bilayer P (90%:45%), to wash fresh semen from 8 men. The % normal morphology (83% 3% vs 81 1%) and motile sperm (74 6% vs 76 3%) did not differ between treatments. Both treatments improved (p<0.09) these parameters over that found for prewashed sperm morphology (72 2%) and motility (62 5%). The % of motile sperm recovered tended to be higher (p=0.1) after SC wash (93 5% vs 80 5%).

Experiment 3: In Expt. 3, semen from 12 men with fertility disorders was evaluated after washing in SC or P. There was no difference in motility of recovered sperm (59 12% vs 60 16%); however, normal membrane function (hypo-osmotic swell test) was better (p=0.045) in SC (70 13% vs 46 10%). Hamster egg SPA was also done on sperm washed from 4 donors in both SC and P. There was no difference in % eggs penetrated (41 2% vs 40 2%) or in the penetration factor (0.48 0.02 vs 0.41 0.03). One man did show a 30% improvement using SC versus P. The SC product is made from a polysaccharide approved for human consumption. It offers a non-toxic one step wash alternative to Percoll.