Gamete Biocompatibility of a Paraben-free Fertility LubricantAshok Agarwal, PhD and Rakesh Sharma, PhD

American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA

Both BD treated and control samples had sperm DFI within the normal range in normozoospermic (DFI - Test 8.8 ± 4.5% vs Control 9.0 ± 4.8%) and oligozoospermic samples (DFI - Test 10.6 ± 3.6% vs Control 10.6 ± 3.5%).

Experiment 2. CASA Total Motile Sperm Count The total motile sperm count (x106) as determined by CASA did not differ significantly between the control (12 ± 4) and BD test samples (11 ± 5) (P > 0.05) motile sperm was comparable (Figure 4).

Experiment 3. Cervical Mucus Penetration TestThe penetration distance traveled by the vanguard sperm into cervical mucus was comparable in both the BD (test) and control samples (Table 1). However, the density of sperm at the furthest measurement in the column was greater for sperm exposed to the BD (P = 0.03)

Experiment 2. Impact of BD on computer-assisted sperm outcomes for washed human sperm. Normospermic sperm samples (n = 11) were incubated for 120 min with or without 10% BD in HTF, followed by computer-assisted semen analysis (CASA) to evaluate Total Motile Sperm Counts (TMC; volume X % motility X concentration). Sperm samples for this aim were prepared on a double density gradient (40% and 90%; Figure 1). Aliquots containing 20 x 106 motile sperm were incubated with HTF with 2% human serum albumin (controls) or 10% BD (test) in HTF with HSA, incubated in a 5% CO2 incubator at 37°C for 2h. Resulting TMC was compared between control and BD treated samples.

Experiment 3. Impact of BD on bull sperm penetration into bovine cervical mucus columns. A sperm penetration assay was done using estrus cow cervical mucus columns and bull sperm. Columns of cervical mucus were exposed to frozen-thawed and washed bull sperm for 30 min in control Tris extender, or with added 10% BD (test). The distance traveled by the vanguard sperm, and the density of sperm at set distances in the columns for each treatment were compared.

Data were analyzed using the Wilcoxon Signed-Rank test for paired data. Data is expressed as mean ± SD.

Introduction

Materials and Methods

Materials and Methods continued Results continued

Experiment 1. Percent Motile Sperm and DNA Fragmentation in Native SemenThere was no difference in average percent progressively motile sperm in the BD test and control sample groups for either the normospermic (Test 72 ± 9% vs Control 73 ± 9%) (Figure 2) or oligospermic (Test 62 ± 3% vs Control 63 ± 3%) groups after 30 min incubation (P > 0.05) (Figure 3).

Results

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Figure 1. Preparation of sperm by double density gradient for CASA studies.

Treatment N CASA Motility

(%) Vanguard

Sperm (cm) Sperm Density

(2 cm) Sperm Density (3 cm)

Control 10 40.0 ± 2.0 5.6 ± 0.2 27.9 ± 1.1 14.4 ± 0.9 *BabyDance 10 42.0 ± 1.0 5.8 ± 0.2 29.7 ± 0.6 16.6 ± 0.6*

*Means differ between treatments at p=0.03

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Figure 4. Total Motile Sperm Count for washed human sperm in Control medium or Baby Dance (Test) samples after 2 hr of incubation at 37°C

Conclusion1. Baby Dance a Paraben-free lubricant did not adversely impact tested sperm function outcomes, including: motility, DNA fragmentation or total motile sperm count, in native semen or washed sperm samples.

2. BD did not affect sperm DNA fragmentation rates or the ability of sperm to penetrate estrus bovine cervical mucus.

3. Baby Dance is the first FDA cleared paraben-free lubricant that is sperm friendly and recommended for Trying-To-Conceive Couples.

Sexual lubricants enhance the pleasure and comfort of intercourse. But in trying-to-conceive couples (TTC), it is critical that lubricants not only enhance the comfort of intimacy, but also that they do not interfere with sperm function. Everyday lubricants harm sperm through: an extremely low pH; a high ion concentration (“hyperosmolar”); and/or inclusion of ingredients that are gamete toxic, making them counterproductive for TTC. In order to identify lubricants that are safe for use by TTC, FDA has created a new fertility lubricant device, Class “PEB”. PEB fertility lubricants require regulatory review of their safety on sperm, eggs and embryos. Such products cannot interfere with clinical semen parameters such as sperm motility, DNA quality and penetration into cervical mucus.

Previously, all FDA cleared PEB fertility lubricants have contained paraben preservatives. Parabens are endocrine disruptors linked to cancer; reproductive, immuno and neuro-toxicity; and mucosal irritation. Additionally, commonly used levels of parabens in lubricants are high enough to interfere with sperm function and in vitro fertilization.

This study was done to evaluate the biocompatibility of a new FDA cleared paraben-free lubricant, “Baby Dance” (BD; Fairhaven Health) on sperm function (e.g. motility, DNA fragmentation and cervical mucus penetrability).

Three biocompatibility studies looking at the impact of BD on sperm function were done, following required institutional approval.

Experiment 1. Impact of BD on motility and DNA fragmentation of human sperm in native semen. Semen samples were obtained from 10 normospermic and 10 oligozoospermic men by masturbation after 72 hrs of abstinence. After liquefaction (20 min at 37°C), samples underwent semen analysis and were assigned to a subject group according to WHO criteria (e.g. normospermic = concentration > 15 X 106 sperm/mL; progressive motility >40%: WHO, 2010). Semen samples were then incubated for 30 min with human tubal fluid (HTF; control group) or HTF with added 10% paraben-free BD lubricant (test group) and evaluated for the percent progressive motility (WHO, 2010).

Sperm DNA Fragmentation Index (% DFI) for sperm in both groups was measured by the sperm chromatin structure assay (SCSA®; Evenson, 2013) via flow cytometry of acridine orange (AO) stained sperm.

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Figure 3. Progressive motility for human sperm in native semen in Control medium or 10% Baby Dance (Test) samples after 30 minutes of incubation at 37°C

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Figure 2. Total Motile Sperm Count for washed human sperm in Control medium or Baby Dance (Test) samples after 2 hr of incubation at 37°C

Table 1. Distance traveled by vanguard sperm exposed to BD.