A Sibling Oocyte Study- Comparison of ZyMotTM Microfluidics Device to Density Gradient for Sperm Selection During ICSI
Phase: NA
Conditions: ART
Sponsor: NYU Langone Health

CLINICAL STUDY PROTOCOL 
 
A Sibling Oocyte Study- Comparison of 
ZyMotTM Microfluidics Device to Density 
Gradient for Sperm Selection During ICSI 
 
Principal Investigator 
Rani Fritz 
 
Sponsor 
DxNow 
401 Professional Drive, Gaithersburg, MD, 20879-3429, United States 
https://zymotfertility.com/ 
 
Protocol Version 
17 March 2021 
version DRAFT 
 
 
 
s21-00021 
NCT04818593

Study: i21-00021 
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Study Personnel 
Principal Investigator 
Rani Fritz 
Email: Rani.Fritz@nyulangone.org 
  
Sub-Investigators 
Alicia Arnott 
Email: Alicia.Arnott@nyulangone.org 
  
  
Meredith Akerman 
Email: Meredith.Akerman@nyulangone.org 
  
  
Linda Sung 
Email: linda.sung@nyulangone.org 
  
  
Maria Saketos 
Email: maria.saketos@nyulangone.org 
  
  
Satu Kuokkanen 
Email: Satu.Kuokkanen@nyulangone.org 
  
  
Vasilios Goudas 
Email: Vasilios.Goudas@nyulangone.org 
  
Jessica Tozour 
Email: Jessica.Tozour@nyulangone.org 
  
  
Daniela Schab 
Email: schabd01@nyulangone.org 
  
  
Catherine Dennis 
Email: Catherine.Dennis@nyulangone.org 
  
Key Study Personnel: 
Alicia Arnott 
Email: Alicia.Arnott@nyulangone.org 
  
  
Meredith Akerman 
Email: Meredith.Akerman@nyulangone.org 
  
 
 
 

Study: i21-00021 
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Synopsis 
 
Primary Objective  
The primary objective of this study is to evaluate whether the percentage of good quality 
embryo formation following ICSI is improved with the use of ZyMot method of microfluidic 
sperm separation compared to density gradient. 
 
Secondary Objectives  
The secondary objectives will be to evaluate: 
1. Sperm parameters prior to and following sperm separation with ZyMot compared to 
density gradient 
2. Normal fertilization rates following ICSI comparing sperm separation with ZyMot with 
density gradient 
3. Euploid rates, in the event that PGT-A was performed, following ICSI comparing 
sperm separation with ZyMot with density gradient 
4. Embryo transfer results including: 
5. Not pregnant 
6. Biochemical pregnancy, defined as BHCG value > 5 mIU/ml without ultrasound 
evidence of gestational sac within the uterus 
7. Clinical miscarriage, defined as loss of a pregnancy following a positive BHCG value 
and ultrasound revealing presence of a gestational sac within the uterus 
8. Clinical pregnancy, defined as positive BHCG with a gestational sac within the uterus 
9. Ongoing Pregnancy, defined as a positive BHCG value with ultrasound evidence of 
intrauterine gestational sac at or past 8 weeks gestation 
10. Live birth defined as birth of a baby at 20 weeks or later, showing signs of life, 
including breathing, beating of the heart, pulsation of the umbilical cord, or definite 
movement of the voluntary muscles. 
11. Ectopic Pregnancy, defined as positive BHCG and presence of pregnancy outside 
of the uterus. 
 
Study Duration 
The study will be completed once 408 oocytes are randomized, which is anticipated to be in 
approximately 1-2 years. 
 
Study Design  
Subjects meeting inclusion criteria will be approached for inclusion into the study and 
informed consent will be obtained from the patient and the sperm source. Controlled ovarian 
hyperstimulation will be performed followed by oocyte retrieval as per our standard protocol. 
On the day of oocyte retrieval, sperm samples will be split into two samples and processed 
via density gradient (our standard sperm separation technique) and ZyMot (our experimental 
arm). Sperm parameters will be evaluated pre and post-sperm separation following ZyMot 
and density gradient. Following oocyte retrieval, the oocytes will be assessed as per our 
standard fashion. In the event that the oocytes will be inseminated via intracytoplasmic 
sperm injection (ICSI) and there are 6 or more mature oocytes, half of the oocytes will be 
inseminated via ZyMot processed sperm and half with sperm processed via density gradient. 
In the event that there are less than 6 mature oocytes or the insemination technique will be 
IVF, the oocytes will only be inseminated with sperm processed via density gradient. 
Additionally, in the event that the sperm sample is insufficient for ZyMot processing (<0.5 
million total motile sperm and/or no sperm observed with progressive forward motility), the 
sperm will not be processed with ZyMot and the patient will be withdrawn from the study.  
 
The primary outcome will be percentage of good quality embryo formation resulting from 
oocytes inseminated following ZyMot separated sperm compared to density gradient 
separated sperm. Secondary outcomes will include percentage of euploid embryos in the 

Study: i21-00021 
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event that PGT-A is performed, pregnancy rates, miscarriage rates, live birth rates, and 
sperm parameters following sperm processing. Decision to transfer an embryo will be made 
as per our standard protocols regardless of the sperm processing technique.  
 
Study Population  
The study population will include patient/sperm donor dyads where all patients over 18 years 
of age are undergoing controlled ovarian hyperstimulation for IVF with ICSI using ejaculate 
sperm and having at least 6 mature oocytes retrieved. The sperm source/donor will provide 
informed consent for the use of their sperm sample in this research study. The relationship 
of the sperm source will can be the intended father, a known sperm donor in a same-sex 
couple or a known donor in the case of a single patient. The identity of the donor cannot be 
anonymous.  
 
Number of Participants  
Following power calculation, 408 oocytes will be needed to randomize (see details in the 
statistics section). The number of patient/sperm source dyads enrolled will depend on the 
number of oocytes obtained from each patient. The maximum number of subjects will be 68 
(in the event that 6 oocytes were obtained from every patient enrolled in the study).  
Number of Study Sites 
This study will take place at NYU Langone Reproductive Specialists of NY. 
 
Primary Outcome Variables  
The primary outcome of our study is to evaluate whether good quality embryo formation is 
significantly improved with ICSI following sperm separation using ZyMot compared to 
density gradient. 
 
Secondary and Exploratory Outcome Variables 
The secondary objectives will be to evaluate: 
1. Sperm parameters prior to and following sperm separation with ZyMot compared to 
density gradient 
2. Normal fertilization rates following ICSI comparing sperm separation with ZyMot with 
density gradient 
3. Euploid rates, in the event that PGT-A was performed, following ICSI comparing 
sperm separation with ZyMot with density gradient 
4. Embryo transfer results including: 
a. Not pregnant 
b. Biochemical pregnancy, defined as BHCG value > 5 mIU/ml without 
ultrasound evidence of gestational sac within the uterus 
c. Clinical miscarriage, defined as loss of a pregnancy following a positive 
BHCG value and ultrasound revealing presence of a gestational sac within 
the uterus 
d. Clinical pregnancy, defined as positive BHCG with a gestational sac within 
the uterus 
e. Ongoing Pregnancy, defined as a positive BHCG value with ultrasound 
evidence of intrauterine gestational sac at or past 8 weeks gestation 
f. 
Live birth, defined as birth of a baby at 20 weeks or later, showing signs of 
life, including breathing, beating of the heart, pulsation of the umbilical cord, 
or definite movement of the voluntary muscles. 
g. Ectopic Pregnancy, defined as positive BHCG and presence of pregnancy 
outside of the uterus. 
 
 
 

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Abbreviations 
Abbreviation  
Explanation 
ART 
Assisted reproductive technologies  
DNA 
Deoxyribonucleic acid  
ICSI 
Intracytoplasmic sperm injection 
IVF 
In vitro fertilization 
PGT 
Preimplantation genetic testing 
PGT-A 
Preimplantation genetic testing for aneuploidy 
PI 
Principle investigator 
RSOFNY 
Reproductive Specialists of New York 
 
 

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Table of Contents 
Synopsis ......................................................................................................................................3 
Primary Objective  ...................................................................................................................3 
Secondary Objectives (if applicable)  ......................................................................................3 
Study Duration  ........................................................................................................................3 
Study Design  ..........................................................................................................................3 
Study Population  ....................................................................................................................4 
Number of Participants  ...........................................................................................................4 
Number of Study Sites.............................................................................................................4 
Primary Outcome Variables  ...................................................................................................4 
Secondary and Exploratory Outcome Variables (if applicable)  .............................................4 
Visit Schedule Table (Optional) ................................................................................................. 
Study Flow Chart (optional) ....................................................................................................... 
Abbreviations ..............................................................................................................................5 
Glossary of Terms ......................................................................................................................... 
1 - Introduction ............................................................................................................................8 
1.1 Introductory Statement  .....................................................................................................8 
2 - Background ............................................................................................................................8 
2.1 Background/prevalence of research topic  .......................................................................8 
2.2.1 Device Preclinical Experience  ...................................................................................9 
2.2.2 Device Clinical Experience  ..................................................................................... 10 
3 - Rationale/Significance ........................................................................................................ 10 
3.1 Problem Statement  ........................................................................................................ 10 
3.2 Purpose of Study/Potential Impact  ................................................................................ 10 
3.3.1 Potential Risks ......................................................................................................... 10 
3.3.2 Potential Benefits  .................................................................................................... 11 
4 - Study Objectives ................................................................................................................. 11 
4.1 Hypothesis ...................................................................................................................... 11 
4.2 Primary Objective  .......................................................................................................... 11 
4.3 Secondary Objectives (if applicable) ............................................................................. 11 
5 - Study Design ...................................................................................................................... 11 
5.1 General Design Description  .......................................................................................... 11 
5.1.1 Study Date Range and Duration .............................................................................. 12 
5.1.2 Number of Study Sites ............................................................................................. 12 
5.2 Outcome Variables ......................................................................................................... 12 
5.2.1 Primary Outcome Variables  .................................................................................... 12 
5.2.2 Secondary and Exploratory Outcome Variables (if applicable)  ............................. 12 
5.3 Study Population  ........................................................................................................... 12 
5.3.1 Number of Participants  ........................................................................................... 13 
5.3.2 Eligibility Criteria/Vulnerable Populations  ............................................................... 13 
6 - Methods .............................................................................................................................. 13 
6.1 Treatment - Device ......................................................................................................... 13 
6.1.1 Intended use for the device ..................................................................................... 14 
6.1.2 Device administration and schedule  ....................................................................... 14 
6.1.3 Method of Assignment/Randomization  ................................................................... 15 
6.1.4 Device Calibration  ................................................................................................... 16 
6.1.5 Storage Condition  ................................................................................................... 16 
6.1.6 Concomittant therapy ............................................................................................... 16 
6.1.7 Restrictions .............................................................................................................. 16 
6.2 Assessments .................................................................................................................. 16 
6.2.1 Efficacy  .................................................................................................................... 16 
6.2.2 Safety/Pregnancy-related policy .............................................................................. 16 
6.2.2.1 Adverse Events Definition and Reporting  ............................................................ 16 
6.2.3 Pharmacokinetics (if applicable) ................................................................................... 

Study: i21-00021 
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6.2.4 Biomarkers (if applicable) ............................................................................................. 
6.3 Study Procedures  ............................................................................................................... 
6.3.1 Study Schedule  ....................................................................................................... 19 
6.3.2 Informed Consent .................................................................................................... 20 
6.3.3 Screening ................................................................................................................. 20 
6.3.4 Recruitment, Enrollment and Retention .................................................................. 20 
6.3.5 On Study Visits ......................................................................................................... 21 
6.3.6 End of Study and Follow-up ..................................................................................... 21 
6.3.7 Removal of subjects  ................................................................................................ 21 
6.4 Statistical Method ........................................................................................................... 21 
6.4.1 Statistical Design ..................................................................................................... 21 
6.4.2 Sample Size Considerations  ................................................................................... 21 
6.4.3 Planned Analyses ........................................................................................................ 22 
6.4.3.1 Primary Analyses  ................................................................................................. 22 
6.4.3.2 Secondary Objectives Analyses  .......................................................................... 22 
6.4.3.3 Safety/Pregnancy-related policy ........................................................................... 22 
6.4.3.4 Analysis of Subject Characteristics ...................................................................... 22 
6.4.3.5 Interim Analysis (if applicable)  ............................................................................. 22 
6.4.3.6 Health economic evaluation .................................................................................. 22 
6.4.3.7 Other ..................................................................................................................... 22 
6.4.4 Subsets and Covariates  .......................................................................................... 22 
6.4.5 Handling of Missing Data  ........................................................................................ 22 
7 - Trial Administration ............................................................................................................. 23 
7.1 Ethical Considerations: Informed Consent/Assent and HIPAA Authorization  .............. 23 
7.2 Institutional Review Board (IRB) Review  ...................................................................... 23 
7.3 Subject Confidentiality  ................................................................................................... 23 
7.4 Deviations/Unanticipated Problems  .............................................................................. 23 
7.5 Data Collection  .............................................................................................................. 23 
7.6 Data Quality Assurance.................................................................................................. 24 
7.7 Study Records ................................................................................................................ 24 
7.8 Access to Source ........................................................................................................... 24 
7.9 Data or Specimen Storage/Security  .............................................................................. 24 
7.10 Retention of Records  ................................................................................................... 25 
7.11 Study Monitoring .......................................................................................................... 25 
7.12 Data Safety Monitoring Plan ........................................................................................ 25 
7.13 Study Modification ........................................................................................................ 25 
7.14 Study Discontinuation ................................................................................................... 25 
7.15 Study Completion  ........................................................................................................ 25 
7.16 Conflict of Interest Policy .............................................................................................. 25 
7.17 Funding Source ............................................................................................................ 25 
7.18 Publication Plan ............................................................................................................ 25 
Appendices ................................................................................................................................... 
List of Tables ................................................................................................................................. 
References .................................................................................................................................... 
 
 
 

Study: i21-00021 
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1 - Introduction 
1.1 Introductory Statement  
The primary objective of this study is to evaluate whether the percentage of good quality embryo 
formation following ICSI is improved with the use of ZyMot method of microfluidic sperm separation 
compared to density gradient. 
 
2 - Background 
2.1 Background/prevalence of research topic  
Sperm integrity and function is critical to embryo formation and IVF success rates. Male factor 
infertility accounts for approximately 1/3 of infertility diagnosis and is the single most common 
diagnosis for IVF cycles in the US. During an IVF cycle, oocytes retrieved are inseminated with sperm 
via IVF or ICSI. IVF is the process by which approximately 50,000-100,000 motile sperm are 
incubated with one or more oocytes, whereas ICSI is the process of selecting and injecting a single 
sperm into an oocyte. ICSI was originally developed to treat male factor infertility, however, its use 
has been greatly expanded in ART including insemination of cryopreserved oocytes, previous 
fertilization failure or poor fertilization results following IVF, unexplained infertility, in vitro maturation, 
cases of PGT when paternal contamination is of concern, and other clinically relevant indications at 
the physician's discretion (1). Selection of healthy sperm for use in ICSI is critical to ICSI success. 
Density gradient is a commonly used method to select sperm for ICSI, which depends on 
centrifugation, and is the standard of care for sperm processing prior to ICSI in our institution. This 
technique requires mechanical and chemical processing that can increase oxygen free radical 
formation, which can cause sperm DNA damage and increase in DNA fragmentation (2, 3). Although 
conflicting data exists as to whether elevated levels of sperm DNA fragmentation compromises live 
birth rates in assisted reproductive technology (ART) treatments, some data has revealed that 
increased sperm DNA fragmentation decreases fertilization rates, decreases progression to good 
quality embryos, decreases pregnancy rates, and increases miscarriage rates (4-10). Studies have 
shown elevated levels of sperm DNA fragmentation in infertile males with abnormal semen 
parameters (11-13), however, interestingly data has revealed that increase levels of DNA 
fragmentation can also be found in infertile males with normal semen parameters (14). 
 
Separation of sperm with microfluidics prior to ART resembles the physiologic environment that sperm 
encounters in the female reproductive tract more closely compared to density gradient, and has been 
proposed to decrease sperm DNA fragmentation. Indeed, separation via ZyMot, a microfluidic 
technique has been shown to significantly decrease sperm DNA fragmentation compared to the 
traditional density gradient technique (15). In a non-randomized study with a small sample size 
enrolling couples with recurrent ART failure and male factor infertility with high sperm 
defragmentation, outcomes with density gradient versus microfluidic sperm separation using ZyMot 
were compared and revealed that embryo euploid rates following PGT-A and pregnancy rates were 
significantly improved with the use of ZyMot (16). A retrospective study evaluating pregnancy rates 
using ZyMot sperm separation compared to density gradient for intrauterine insemination (IUI) 
revealed significantly higher ongoing pregnancy rates with use of ZyMot compared to density gradient 
(17). Additionally, a prospective randomized study evaluating ZyMot sperm separation with the swim 
up technique for patients undergoing ICSI due to unexplained infertility revealed a significant increase 
in good quality embryo formation with ZyMot sperm separation, however, this study included a 
significant proportion of embryos evaluated and transferred at day 3 or 4 as opposed to day 5, which 
is our standard of care (18). 
 
The aim of our study is to evaluate whether good quality embryo formation is significantly different 
following ICSI comparing sperm separated using ZyMot with density gradient. 
 
References 

Study: i21-00021 
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1. Practice Committees of the American Society for Reproductive M, the Society for Assisted 
Reproductive Technology. Electronic address aao. Intracytoplasmic sperm injection (ICSI) for 
non-male factor indications: a committee opinion. Fertility and Sterility 2020;114:239-45. 
2. Zalata A, Hafez T, Comhaire F. Evaluation of the role of reactive oxygen species in male 
infertility. Human Reproduction 1995;10:1444-51. 
3. Zini A, Finelli A, Phang D, Jarvi K. Influence of semen processing technique on human sperm 
DNA integrity. Urology 2000;56:1081-4. 
4. Borini A, Tarozzi N, Bizzaro D, Bonu MA, Fava L, Flamigni C et al. Sperm DNA fragmentation: 
paternal effect on early post-implantation embryo development in ART. Human Reproduction 
2006;21:2876-81. 
5. Seli E, Gardner DK, Schoolcraft WB, Moffatt O, Sakkas D. Extent of nuclear DNA damage in 
ejaculated spermatozoa impacts on blastocyst development after in vitro fertilization. Fertility 
and Sterility 2004;82:378-83. 
6. Robinson L, Gallos ID, Conner SJ, Rajkhowa M, Miller D, Lewis S et al. The effect of sperm 
DNA fragmentation on miscarriage rates: a systematic review and meta-analysis. Human 
Reproduction 2012;27:2908-17. 
7. McQueen DB, Zhang J, Robins JC. Sperm DNA fragmentation and recurrent pregnancy loss: 
a systematic review and meta-analysis. Fertility and Sterility 2019;112:54-60 e3. 
8. Borges E, Jr., Zanetti BF, Setti AS, Braga D, Provenza RR, Iaconelli A, Jr. Sperm DNA 
fragmentation is correlated with poor embryo development, lower implantation rate, and higher 
miscarriage rate in reproductive cycles of non-male factor infertility. Fertility and Sterility 
2019;112:483-90. 
9. Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G. Sperm chromatin damage 
impairs human fertility. The Danish First Pregnancy Planner Study Team. Fertility and Sterility 
2000;73:43-50. 
10. Bungum M, Humaidan P, Axmon A, Spano M, Bungum L, Erenpreiss J et al. Sperm DNA 
integrity assessment in prediction of assisted reproduction technology outcome. Human 
Reproduction 2007;22:174-9. 
11. Zini A, Bielecki R, Phang D, Zenzes MT. Correlations between two markers of sperm DNA 
integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men. Fertility and 
Sterility 2001;75:674-7. 
12. Huang CC, Lin DP, Tsao HM, Cheng TC, Liu CH, Lee MS. Sperm DNA fragmentation 
negatively correlates with velocity and fertilization rates but might not affect pregnancy rates. 
Fertility and Sterility 2005;84:130-40. 
13. Moskovtsev SI, Willis J, White J, Mullen JB. Sperm DNA damage: correlation to severity of 
semen abnormalities. Urology 2009;74:789-93. 
14. Saleh RA, Agarwal A, Nelson DR, Nada EA, El-Tonsy MH, Alvarez JG et al. Increased sperm 
nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertility and 
Sterility 2002;78:313-8. 
15. Quinn MM, Jalalian L, Ribeiro S, Ona K, Demirci U, Cedars MI et al. Microfluidic sorting selects 
sperm for clinical use with reduced DNA damage compared to density gradient centrifugation 
with swim-up in split semen samples. Human Reproduction 2018;33:1388-93. 
16. Parrella A, Keating D, Cheung S, Xie P, Stewart JD, Rosenwaks Z et al. A treatment approach 
for couples with disrupted sperm DNA integrity and recurrent ART failure. Journal of assisted 
reproduction and genetics 2019;36:2057-66. 
17. Gode F, Bodur T, Gunturkun F, Gurbuz AS, Tamer B, Pala I et al. Comparison of microfluid 
sperm sorting chip and density gradient methods for use in intrauterine insemination cycles. 
Fertility and Sterility 2019;112:842-8 e1. 
18. Yetkinel S, Kilicdag EB, Aytac PC, Haydardedeoglu B, Simsek E, Cok T. Effects of the 
microfluidic chip technique in sperm selection for intracytoplasmic sperm injection for 
unexplained infertility: a prospective, randomized controlled trial. Journal of assisted 
reproduction and genetics 2019;36:403-9. 
 
2.2.1 Device Preclinical Experience  

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The ZyMot Multi Sperm Separation Device (850μl) is used to prepare motile sperm for use in ART. 
ZyMot separates motile sperm from the semen based on the motility of the sperm within a 
microenvironment. The ZyMot Multi sperm separation device has an inlet port for applying the semen 
sample to the lower sample chamber and an outlet port for collecting the motile sperm from the upper 
collection chamber. The chambers are separated by a microporous filter or membrane. After 
incubation at 37C for 30 min, the culture medium containing motile sperm is collected from the outlet 
port. 
 
The device is made from Polymethylmethacrylate, borosilicate glass, flash-spun high-density 
polyethylene fibers. The devices are gamma radiation-sterilized devices with a sterility assurance 
level (SAL) of 10-6. They are individually packaged and for single-use only. 
 
This device has been FDA approved for our proposed use (see attached). 
 
2.2.2 Device Clinical Experience  
Separation via ZyMot has been shown to significantly decrease sperm DNA fragmentation compared 
to the traditional density gradient technique (15). In a non-randomized study with a small sample size 
enrolling couples with recurrent ART failure and male factor infertility with high sperm 
defragmentation, outcomes with density gradient versus microfluidic sperm separation using ZyMot 
were compared and revealed that embryo euploid rates following PGT-A and pregnancy rates were 
significantly improved with the use of ZyMot (16). A retrospective study evaluating pregnancy rates 
using ZyMot sperm separation compared to density gradient for intrauterine insemination (IUI) 
revealed significantly higher ongoing pregnancy rates with use of ZyMot compared to density gradient 
(17). Additionally, a prospective randomized study evaluating ZyMot sperm separation with the swim 
up technique for patients undergoing ICSI due to unexplained infertility revealed a significant increase 
in good quality embryo formation with ZyMot sperm separation, however, this study included a 
significant proportion of embryos evaluated and transferred at day 3 or 4 as opposed to day 5, which 
is our standard of care (18). 
 
3 - Rationale/Significance 
3.1 Problem Statement  
Commonly used methods for separating and processing sperm prior to ICSI involves mechanical and 
chemical processing such as density gradient separation. Density gradient sperm separation can 
increase sperm oxygen free radicals and DNA fragmentation. Sperm oxygen free radicals and DNA 
fragmentation can adversely affect sperm quality which can adversely affect ART success rates. 
Sperm separation via microfluidics separates sperm in a more physiologic fashion and has been 
shown to decrease DNA fragmentation as compared to the density gradient. 
 
3.2 Purpose of Study/Potential Impact  
We intend to evaluate whether formation of high-quality embryos following ICSI are improved with 
ZyMot microfluidic sperm separation compared to density gradient. Potential impact of the study could 
be that good quality embryo formation is improved with ZyMot compared with density gradient, which 
could have a positive impact on ART success rates. 
 
3.3.1 Potential Risks  
Potential risks of the study include the possibility that good quality embryo formation may not be 
improved with the use of ZyMot or may be inferior compared to density gradient. Inferior embryos 
could negatively impact the ability of the embryo to lead to yield a liveborn fetus. To our knowledge, 
existing literature has not revealed any detrimental effect to the quality of embryo formation with the 
use of the ZyMot device. Additionally, enrollment in the study can incur possible psychological distress 
due to study participation. 
 

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Procedures to limit psychological distress will be a thorough informed consent process, ability to 
discuss with any participating provider questions related to the study, and ability to withdraw 
enrollment at any point prior to insemination of the oocytes. 
 
3.3.2 Potential Benefits  
Potential benefit to study enrollment could be that good quality embryo formation is improved following 
sperm separation with ZyMot compared to density gradient, which could improve the participant's 
ART success rates. 
 
4 - Study Objectives 
4.1 Hypothesis 
We hypothesize that the percentage of good quality embryo formation following ICSI will be improved 
with sperm separated via ZyMot compared to density gradient. 
 
4.2 Primary Objective  
The primary objective of this study is to evaluate whether the percentage of good quality embryo 
formation following ICSI is improved with the use of ZyMot method of microfluidic sperm separation 
compared to density gradient. 
 
4.3 Secondary Objectives  
The primary objective of this study is to evaluate whether the percentage of good quality embryo 
formation following ICSI is improved with the use of ZyMot method of microfluidic sperm separation 
compared to density gradient. 
 
The secondary objectives will be to evaluate: 
1. Sperm parameters prior to and following sperm separation with ZyMot compared to density 
gradient 
2. Normal fertilization rates following ICSI comparing sperm separation with ZyMot with density 
gradient 
3. Euploid rates, in the event that PGT-A was performed, following ICSI comparing sperm 
separation with ZyMot with density gradient 
4. Embryo transfer results including: 
a. Not pregnant 
b. Biochemical pregnancy, defined as BHCG value > 5 mIU/ml without ultrasound 
evidence of gestational sac within the uterus 
c. Clinical miscarriage, defined as loss of a pregnancy following a positive BHCG value 
and ultrasound revealing presence of a gestational sac within the uterus 
d. Clinical pregnancy, defined as positive BHCG with a gestational sac within the uterus 
e. Ongoing Pregnancy, defined as a positive BHCG value with ultrasound evidence of 
intrauterine gestational sac at or past 8 weeks gestation 
f. 
Live birth defined as birth of a baby at 20 weeks or later, showing signs of life, including 
breathing, beating of the heart, pulsation of the umbilical cord, or definite movement of 
the voluntary muscles. 
g. Ectopic Pregnancy, defined as positive BHCG and presence of pregnancy outside of 
the uterus. 
 
As per our standard of care, patients will be contacted by embryology staff via phone after their 
expected due to date for their pregnancy outcome. Pregnancy outcomes will be recorded in the 
patient’s medical record.  
 
5 - Study Design 
5.1 General Design Description  

Study: i21-00021 
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This study will be a randomized sister oocyte (oocytes from the same patient within the same cycle) 
study at a single academic institution. By randomizing sister oocytes, we will eliminate any potential 
confounding variables. 
 
5.1.1 Study Date Range and Duration 
The anticipated study duration is anticipated to be approximately be 1-2 years following initiation of 
subject recruitment.  
 
5.1.2 Number of Study Sites 
This study will take place at NYU Langone Reproductive Specialists of NY. 
 
5.2 Outcome Variables 
 
5.2.1 Primary Outcome Variables  
The primary objective of this study is to evaluate whether the percentage of good quality embryo 
formation following ICSI is improved with the use of ZyMot method of microfluidic sperm separation 
compared to density gradient. 
 
Good quality embryos will be defined as blastocyst stage embryos on day 5 or day 6 of culture with 
an overall quality grade of good or fair. 
 
5.2.2 Secondary and Exploratory Outcome Variables 
The secondary outcomes will be to evaluate: 
1. Sperm parameters prior and following sperm separation with ZyMot compared to density 
gradient. 
2. Sperm parameters that will be evaluated are sperm concentration, percent motility, and grade 
of forward progression. 
3. Normal fertilization rates following ICSI comparing sperm separation with ZyMot to density 
gradient. Normal fertilization will be defined as visualization of exactly two pronuclei between 
16-18 hours post-insemination. 
4. Euploid, aneuploid, and mosaic rates in the event that PGT-A was performed following ICSI 
comparing sperm separation with ZyMot to density gradient. PGT-A biopsy samples will be 
sent to and analyzed by Cooper Genomics. 
5. Euploid embryo will be defined as 46XX or 46XY. Mosaic rates will be defined as presence of 
euploid and aneuploid cells and be reported as high or low mosaic. High mosaicism will be 
defined as embryos having 41 - 80% aneuploid cells, whereas, low mosaicism will be defined 
as 20 - 40% aneuploid cells. 
6. Embryo transfer results including: 
a. Not pregnant 
b. Biochemical pregnancy, defined as BHCG value > 5 mIU/ml without ultrasound 
evidence of gestational sac within the uterus 
c. Clinical miscarriage, defined as loss of a pregnancy following a positive BHCG value 
and ultrasound revealing presence of a gestational sac within the uterus 
d. Clinical pregnancy, defined as positive BHCG with a gestational sac within the uterus 
e. Ongoing Pregnancy, defined as a positive BHCG value with ultrasound evidence of 
intrauterine gestational sac at or past 8 weeks gestation 
f. 
Live birth defined as birth of a baby at 20 weeks or later, showing signs of life, including 
breathing, beating of the heart, pulsation of the umbilical cord, or definite movement of 
the voluntary muscles. 
g. Ectopic Pregnancy, defined as positive BHCG and presence of pregnancy outside of 
the uterus. 
 
5.3 Study Population  

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The study population will include all patient/sperm source dyads over 18 years of age undergoing 
controlled ovarian hyperstimulation for IVF with ICSI using ejaculate sperm from known sperm source 
and having at least 6 mature oocytes retrieved. 
 
5.3.1 Number of Participants  
As this study will be randomizing oocytes, and therefore the number of participants will depend on the 
average number of oocytes retrieved per patient. Following power calculation,408 oocytes are needed 
for randomization in order to detect a significant difference in our primary outcome. If 6 oocytes are 
obtained from each patient enrolled in the study, then 68 patient/sperm source dyads will be enrolled. 
 
Based on our 2020 data, there was a total of 1,680 mature oocytes from 137 retrievals that produced 
6 or more mature oocytes for ICSI insemination, from 121 different patients. The average number of 
mature oocytes within ICSI cycles with 6 or more mature oocytes obtained was 12 mature oocytes. 
Based on this data, we anticipate 138 patient cycles are needed for this study and this could include 
up to 138 patient/sperm source dyads. 
  
5.3.2 Eligibility Criteria/Vulnerable Populations  
Eligibility will be determined by treating physician, nurse practitioner, or research assistant involved 
in the care of the subject. 
 
Inclusion criteria for patients will include: 
1. Patient(s) over 18 years of age 
2. Patient(s) capable of providing informed consent 
3. Use or possible use of ICSI for oocyte insemination 
4. At least 6 mature oocytes at time of insemination via ICSI 
 
Exclusion criteria for patients: 
1. Patient under 18 y/o 
2. Patients not capable of providing informed consent 
3. Use of IVF for insemination 
4. Less than 6 mature oocytes at time of retrieval  
5. Anonymous donor sperm source 
6. Surgically retrieved sperm 
7. Sperm sample not sufficient for use with ZyMot device 
 
Inclusion criteria for donors: 
1. Donor(s) over 18 years of age 
2. Donor(s) capable of providing informed consent 
3. Use of ejaculate sperm, fresh or frozen, for insemination 
4. Sufficient sperm for use of ZyMot 
 
Exclusion criteria for donors: 
1. Anonymous donors 
 
This study will not enroll vulnerable subjects; however, we routinely follow patients through their 
pregnancy to determine the outcome of embryo-transfer and will use the data from their standard of 
care follow-up calls for this study. The follow-up is considered minimal risk as it only involves data 
collection. No inducements, monetary or otherwise, will be offered to terminate a pregnancy. 
Individuals engaged in the research will have no part in any decisions as to the timing, method, or 
procedures used to terminate a pregnancy. Individuals engaged in the research will have no part in 
determining the viability of a neonate. 
 
6 - Methods 
6.1 Treatment – Device 

Study: i21-00021 
Page 14 of 25 
 
6.1.1 Intended use for the device 
The intended use for the ZyMot device is to separate sperm prior to ICSI insemination alongside and 
comparing it to sperm separation with use of density gradient.  
 
6.1.2 Device administration and schedule  
Patient/sperm source dyads enrolled in the study and meeting inclusion criteria will have their sperm 
sample prepared prior to, or near the time of, oocyte retrieval. Semen parameters will be recorded 
prior to separation. Semen will be split and separated via ZyMot and density gradient. 
 
Density gradient will be performed as follows: 
• 
Density gradient centrifugation will be performed using a one-layer preparation of 90% Isolate 
(Irvine Scientific) in 15 mL conical tubes. Semen will be layered over 1 mL of gradient and 
then centrifuged for 15 min at 300xg. The supernatant will be removed and discarded. The 
sperm pellet will be washed by mixing with Multipurpose Handling Medium Complete (Irvine 
Scientific) and centrifuging the sample for 5 min at 400xg. After the wash, the supernatant is 
removed and discarded and the pellet is re-suspended in culture medium (Continuous Single 
Culture-NX Complete, Irvine Scientific), assessed for sperm parameters, and held at room 
temperature until insemination. 
 
ZyMot separation using the Multi (850 uL) microfluidic separation device will be performed as 
follows: 
• 
850 uL of untreated semen will be directly deposited into the inlet port of the ZyMotTM Multi 
device, followed by placement of 750 uL culture medium (Continuous Single Culture- NX 
complete, Irvine Scientific) in the outlet port and throughout the upper collection chamber. The 
device will then be incubated in a humidified 37C CO2 incubator for 30 minutes. During 
incubation, the healthiest and most motile sperm will swim through the microporous filter and 
into the upper collection chamber, where they will be recovered via the outlet port. 500 uL of 
the sperm sample will be removed and placed in a separate tube for analysis and 
insemination. 
• 
After sperm processing, sperm parameters will be evaluated and recorded for each 
preparation, including sperm concentration, percent motility, and grade of forward 
progression. The density gradient preparation results will be recorded as Sample 1 in the 
electronic medical record (eMR) system gamete sheet. The ZyMot preparation results will be 
recorded as Sample 2 in the eMR system gamete sheet. 
• 
Mode of oocyte insemination is ordered by the physician of record in the patient’s chart. In the 
event that ICSI is ordered, the oocytes will be inseminated via ICSI. In the event that the 
physician orders ICSI per embryologist, the decision to perform ICSI will be a clinical decision 
based on the final prepared semen sample and/or other relevant clinical data. The sample 
must meet the following minimal criteria below in order to be eligible for IVF: 
 
 
 
 
 
 
 
 
• 
In the event that any of the parameters above are not met and/or other clinical parameters 
favoring ICSI insemination over IVF including, but not limited to, microscopic appearance of 
the oocytes, review of previous insemination cycles, and previous morphology grading of 
sperm, ICSI will be performed.  
• 
In the event that the parameters above are met and there are not any other factors favoring 
ICSI insemination, the oocytes will be inseminated via IVF from sperm separated by density 
Parameters 
Initial Sample 
Final Sample 
Volume 
≥ 1.0 mL 
≥ 0.25 mL 
Concentration 
≥ 15 mil/mL 
NA 
Motility 
≥ 35% 
≥ 70% 
Forward 
Progression 
 
≥ 2 
 
≥ 3 

Study: i21-00021 
Page 15 of 25 
gradient and the oocytes will not be inseminated via ZyMot separated sperm, however, the 
pre and post sperm parameters following ZyMot separated sperm and density gradient 
separated sperm will be recorded as per our secondary objectives. Similarly, in the event that 
there are less than 6 mature oocytes, the oocytes will only be inseminated via density gradient 
separated sperm, however, pre and post sperm parameters following ZyMot separated sperm 
and density gradient separated sperm will be recorded as per our secondary objectives.  
• 
As per our standard verbal and written consenting protocols, patients are aware that their 
oocytes may undergo ICSI the day of oocyte retrieval based on above criteria. The decision 
to perform ICSI will not be affected in any way by enrollment into this study. 
• 
Following oocyte retrieval, oocytes will be denuded of their cumulus cells per standard 
protocol. If at least 6 mature oocytes (determined by visualization of a polar body) are present, 
half the oocytes will be ICSI inseminated with sperm prepared via ZyMot device and half will 
be ICSI inseminated with sperm prepared with density gradient. Patients whose patient 
identification number (PID) is even will have the group of oocytes inseminated with sperm 
separated via ZyMot device recorded first in the eMR gamete sheet (beginning with oocyte 
#1.) Patients with an odd PID will have the oocytes inseminated with sperm separated via 
density gradient recorded first in the eMR gamete sheet (beginning with oocyte #1.) In the 
event of an odd number of oocytes, the patients with even PIDs will have the extra oocyte 
inseminated with sperm separated via Zymot device and patients with odd PIDs will have the 
extra oocyte inseminated with sperm separated via density gradient. Inseminated oocytes will 
be grouped into separate wells of a LifeGlobal 4 well GPS dish (Cooper Surgical) by the sperm 
separation method — one well will contain all oocytes inseminated with sperm selected by 
ZyMot device and a separate well will contain all oocytes inseminated with sperm selected by 
density gradient centrifugation. 
 
Control Group of Oocytes: 
Half of the eggs will be inseminated via ICSI using sperm selected by conventional gradient 
centrifugation. 
 
Treatment Group of Oocytes: 
• 
Half of the eggs will be inseminated via ICSI using sperm selected using ZyMot. 
• 
Fertilization assessment will be performed between 16 and 18 hours after insemination and 
normal fertilization will be considered visualization of exactly two pronuclei. Normally fertilized 
oocytes (embryos) will then be cultured in Continuous Single Culture Medium — NX Complete 
(Irvine Scientific) in separate, numbered droplets in LifeGlobal microdrop GPS dishes (Cooper 
Surgical.) The numbers of the droplets will correlate to oocyte/embryo number in the eMR 
gamete sheet and, thereby, also correlate to the type of sperm used for the insemination. 
Development of the embryos will be assessed on culture days 5 and 6 and embryo stage and 
grade will be recorded for each embryo. 
• 
Decision as to which embryo to transfer will be made by the clinical team irrespective of type 
of sperm preparation for insemination. Transfer of embryo is generally based on ploidy status 
(if PGT-A was performed) or blastocyst grade (if PGT-A was not performed). In the event that 
two embryos of similar quality are available for transfer, priority is given to lowest embryo 
number (correlated to oocyte/embryo number). The decision as to which embryo to transfer 
will be made regardless of whether the sperm used to inseminate the oocyte was processed 
with density gradient or ZyMot and will be as per our standard protocol.  
 
6.1.3 Method of Assignment/Randomization  
Patient/sperm source dyads meeting inclusion criteria and enrolled in the study will have their sperm 
sample prepared prior to or near the time of oocyte retrieval. Semen parameters will be recorded prior 
to separation. Semen will be split and separated via ZyMot and density gradient. 
 
Following oocyte retrieval, oocytes will be denuded of their cumulus cells per standard protocol. If at 
least 6 mature oocytes (determined by visualization of a polar body) are present, half the oocytes will 

Study: i21-00021 
Page 16 of 25 
be ICSI inseminated with sperm separated via ZyMot device and half will be ICSI inseminated with 
sperm prepared with density gradient. Patients whose patient identification number (PID) is even will 
have the group of oocytes inseminated with sperm separated via ZyMot device recorded first in the 
eMR gamete sheet (beginning with oocyte #1.) Patients with an odd PID will have the oocytes 
inseminated with sperm separated via density gradient recorded first in the eMR gamete sheet 
(beginning with oocyte #1.) In the event of an odd number of oocytes, the patients with even PIDs will 
have the extra oocyte inseminated with sperm separated via Zymot device and patients with odd PIDs 
will have the extra oocyte inseminated with sperm separated via density gradient. 
 
6.1.4 Device Calibration  
No calibration of the device is required. Each device is single use and disposable after use. 
 
6.1.5 Storage Condition  
The device will be stored at room temperature within our embryology laboratory as per manufacturer 
protocol. Receipt of the device, including number of devices received, lot number(s) of the devices, 
open and close dates for each lot, and certificates of analysis for each lot will be recorded and/or filed. 
Unused devices at the conclusion of the study will be returned to the study sponsor. 
 
6.1.6 Concomitant therapy 
None 
 
6.1.7 Restrictions  
Restrictions for use of the device will include severe male factor defined as an initial ejaculate sample 
with <0.5 million total motile sperm and/or no sperm observed with progressive forward motility 
(forward progression grade of 2.) Any surgically derived sperm will be excluded from the study. 
Additionally, when orders are written for ICSI per embryologist, any specimen not meeting above 
criteria for ICSI (see section 6.1.2), will be inseminated via IVF and therefore, their oocytes will not be 
inseminated with ZyMot separated sperm. 
 
6.2 Assessments 
 
6.2.1 Efficacy  
The efficacy of the device will be evaluated by our primary objective, percentage of good quality 
embryos available following ICSI insemination with ZyMot separated sperm compared to insemination 
with density gradient separated sperm.  
 
6.2.2 Safety/Pregnancy-related policy 
Not Applicable. 
 
6.2.2.1 Adverse Events Definition and Reporting  
Definitions 
Unanticipated Problems Involving Risk to Subjects or Others Any incident, experience, or outcome 
that meets all of the following criteria: Unexpected in nature, severity, or frequency (i.e. not described 
in study-related documents such as the IRB-approved protocol or consent form, the investigators 
brochure, etc.) 
 
Related or possibly related to participation in the research (i.e. possibly related means there is a 
reasonable possibility that the incident experience, or outcome may have been caused by the 
procedures involved in the research) 
 
Suggests that the research places subjects or others at greater risk of harm (including physical, 
psychological, economic, or social harm). 
 
Adverse Event 

Study: i21-00021 
Page 17 of 25 
An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in 
severity during the course of the study. Intercurrent illnesses or injuries should be regarded as 
adverse events. Abnormal results of diagnostic procedures are considered adverse events if the 
abnormality: 
• 
results in study withdrawal 
• 
is associated with a serious adverse event 
• 
is associated with clinical signs or symptoms 
• 
leads to additional treatment or to further diagnostic tests 
• 
is considered by the investigator to be of clinical significance 
 
Serious Adverse Event 
Adverse events are classified as serious or non-serious. A serious adverse event is any AE 
that is: 
• 
fatal 
• 
life-threatening 
• 
requires or prolongs hospital stay 
• 
results in persistent or significant disability or incapacity 
• 
a congenital anomaly or birth defect 
• 
an important medical event 
• 
Important medical events are those that may not be immediately life threatening, but are 
clearly of major clinical significance. They may jeopardize the subject, and may require 
intervention to prevent one of the other serious outcomes noted above. For example, drug 
overdose or abuse, a seizure that did not result in in-patient hospitalization or intensive 
treatment of bronchospasm in an emergency department would typically be considered 
serious. 
 
All adverse events that do not meet any of the criteria for serious should be regarded as non-serious 
adverse events. 
 
Preexisting Condition 
A preexisting condition is one that is present at the start of the study. A preexisting condition should 
be recorded as an adverse event if the frequency, intensity, or the character of the condition worsens 
during the study period. 
 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting condition. At 
the end of the study, any new clinically significant findings/abnormalities that meet the definition of an 
adverse event must also be recorded and documented as an adverse event. 
 
Post-study Adverse Event 
All unresolved adverse events should be followed by the investigator until the events are resolved, 
the subject is lost to follow-up, or the adverse event is otherwise explained. At the last scheduled visit, 
the investigator should instruct each subject to report any subsequent event(s) that the subject, or the 
subject's personal physician, believes might reasonably be related to participation in this study. The 
investigator should notify the study sponsor of any death or adverse event occurring at any time after 
a subject has discontinued or terminated study participation that may reasonably be related to this 
study. The sponsor should also be notified if the investigator should become aware of the 
development of cancer or of a congenital anomaly in a subsequently conceived offspring of a subject 
that has participated in this study. 
 
 Recording of Adverse Events 
At each contact with the subject, the investigator must seek information on adverse events by specific 
questioning and, as appropriate, by examination. Information on all adverse events should be 
recorded immediately in the source document, and also in the appropriate adverse event module of 

Study: i21-00021 
Page 18 of 25 
the case report form. All clearly related signs, symptoms, and abnormal diagnostic procedures results 
should recorded in the source document, though should be grouped under one diagnosis. 
 
All adverse events occurring during the study period must be recorded. The clinical course of each 
event should be followed until resolution, stabilization, or until it has been determined that the study 
treatment or participation is not the cause. Serious adverse events that are still ongoing at the end of 
the study period must be followed up to determine the final outcome. Any serious adverse event that 
occurs after the study period and is considered to be possibly related to study participation should be 
recorded and reported immediately. 
 
Reporting of Serious Adverse Events and Unanticipated Problems 
 
For Narrative Reports of Safety Events 
 
If the report is supplied as a narrative, the minimum necessary information to be provided at the time 
of the initial report includes: 
• 
Study identifier 
• 
Study Center 
• 
Subject number 
• 
A description of the event 
• 
Date of onset 
• 
Current status 
• 
Whether study treatment was discontinued 
• 
The reason why the event is classified as serious 
• 
Investigator assessment of the association between 
the event and study treatment 
  
Investigator reporting: notifying the IRB Federal regulations require timely reporting by investigators 
to their local IRB of unanticipated problems posing risks to subjects or others. The following describes 
the NYULMC IRB reporting requirements, though Investigators at participating sites are responsible 
for meeting the specific requirements of their IRB of record. 
 
Report promptly, but no later than 5 working days: 
Researchers are required to submit reports of the following problems promptly but no later than 5 
working days from the time the investigator becomes aware of the event: 
 
Unanticipated problems including adverse events that are unexpected and related 
Unexpected: An event is "unexpected" when its specificity and severity are not accurately reflected 
in the protocol-related documents, such as the IRB-approved research protocol, any applicable 
investigator brochure, and the current IRB-approved informed consent document and other relevant 
sources of information, such as product labeling and package inserts. 
 
Related to the research procedures: An event is related to the research procedures if in the opinion 
of the principal investigator or sponsor, the event was more likely than not to be caused by the 
research procedures. 
 
Harmful: either caused harm to subjects or others, or placed them at increased risk 
 
Other Reportable events: The following events also require prompt reporting to the IRB, though no 
later than 5 working days: 
 
Complaint of a research subject when the complaint indicates unexpected risks or the complaint 
cannot be resolved by the research team. 
 
Protocol deviations or violations (includes intentional and accidental/unintentional deviations from 
the IRB approved protocol) for any of the following situations: 
one or more participants were placed at increased risk of harm 
the event has the potential to occur again 
the deviation was necessary to protect a subject from immediate harm 

Study: i21-00021 
Page 19 of 25 
Breach of confidentiality 
 
New Information indicating a change to the risks or potential benefits of the research, in terms of 
severity or frequency. (e.g. analysis indicates lower-than-expected response rate or a more severe or 
frequent side effect; FDA labeling change or withdrawal from market) 
 
Reporting Process 
The reportable events noted above will be reported to the IRB using a Reportable New Information 
submission and will include a description of the event with information regarding its fulfillment of the 
above criteria, follow-up/resolution, and need for revision to consent form and/or other study 
documentation. Copies of each report and documentation of IRB notification and receipt will be kept 
in the Clinical Investigator's study file. 
 
6.3.1 Study Schedule  
Subject time devoted to this study will be the reviewing and signing the informed consent. 
 
The timepoints for involvement in the study will be: 
1. The process of informed consent for patient/sperm source dyads 
2. The oocyte retrieval- After retrieval in the event that there are 6 or more mature oocytes, the 
couple will be enrolled into the study 
3. The sperm separation- The sample will either be obtained via fresh production from sperm 
source the day of oocyte retrieval or will be cryopreserved sperm. The sample will be assessed 
the day of oocyte retrieval and if the sperm sample is sufficient, it will be processed via ZyMot 
and density gradient. Following processing of the sperm sample, in the event that there are 6 
mature oocytes available to inseminate, the patient will be enrolled into the study. Oocytes will 
be randomized to insemination via density gradient separated sperm and ZyMot separated 
sperm. 
4. In the event that the orders were written for ICSI per embryologist, if the patient does not meet 
criteria for IVF the day of oocyte retrieval (as per section 6.3), the oocytes will be inseminated 
via IVF with density separated sperm only, however, the pre and post sperm parameters 
following ZyMot separated sperm and density gradient separated sperm will be recorded as 
per our secondary objectives. Similarly, in the event that there are less than 6 mature oocytes, 
the oocytes will only be inseminated via density gradient separated sperm, however, pre and 
post sperm parameters following ZyMot separated sperm and density gradient separated 
sperm will be recorded as per our secondary objectives. Insemination- Following oocyte 
retrieval, those enrolled into the study will have their oocytes split and inseminated with ZyMot 
separated sperm and density gradient separated sperm.  
5. Fertilization check- Approximately 16-18 hours following insemination the embryos will be 
assessed for fertilization.  
6. Five and 6 days following insemination, the embryos will be assessed for stage of growth and 
those reaching blastocyst stage will be given a morphological grade.  
7. In the event that patient will undergo a fresh transfer the patient will be transferred 5 days 
following oocyte retrieval. In the event that pre-implantation genetic testing is performed or the 
embryos are cryopreserved for any other reason, the patient may undergo a frozen embryo 
transfer in standard fashion. 
8. Nine days following embryo transfer, the patient will follow up for a pregnancy test. In the event 
its is positive, the patient will follow up approximately 2-3 days later for a repeat pregnancy 
test. Depending on the value, the patient will either follow up again for a pregnancy test or 
follow up approximately 1 week later for an ultrasound. In the event of a normal intrauterine 
pregnancy, the patient is generally referred to her Obstetrician for obstetric care.  
 
As per our standard of care, patients will be contacted by embryology staff via phone after their 
expected due to date for their pregnancy outcome. Pregnancy outcomes will be recorded in the 
patient’s medical record.  
 

Study: i21-00021 
Page 20 of 25 
6.3.2 Informed Consent  
Consent and other informational documents provided to participants consent forms describing in 
detail the study agent, study procedures, and risks are given to the participant and written 
documentation of informed consent is required prior to starting intervention/administering study 
product. 
 
Consent procedures and documentation informed consent is a process that is initiated prior to the 
individual's agreeing to participate in the study and continues throughout the individual's study 
participation. Extensive discussion of risks and possible benefits of participation will be provided to 
the participants. Consent forms will be IRB-approved and the participant will be asked to read and 
review the document. All discussions about this research with patient/sperm source dyads will occur 
in private consultation offices, exam rooms, or a secure WebEx platform. The patient/sperm source 
dyad will have the consent emailed to the patient/sperm source dyad to be signed during the WebEx 
meeting or in person. The study investigator and or research assistant will explain the study protocol 
and details of the patient/sperm source’s role and anticipated experience during the procedure, 
allowing the patient/sperm sources to ask questions, which will be answered to the subject's 
satisfaction. The consenter will also ask questions of the prospective patient/sperm sources to assess 
that they have understood the information presented to them. The participants should have the 
opportunity to discuss the study with their relatives or think about it prior to agreeing to participate. 
The patient/sperm sources will sign the informed consent document prior to any procedures being 
done specifically for the study. The investigator or research assistant who obtained consent will sign 
the consent form. A signed copy of the consent form will be provided to the patient/sperm source 
dyad. The day of the oocyte retrieval, the sperm will be processed as explained above and used to 
inseminate the occytes with both sperm processed using the density gradient and ZyMot microfluidics 
as long as they are eligible for inclusion. Subjects can withdraw from the study at any time if they no 
longer wish their data to be collected and used for study purposes. However, they will be informed 
that if they withdraw after insemination, the insemination process cannot be reversed. The rights and 
welfare of the participants will be protected by emphasizing to them that the quality of their medical 
care will not be adversely affected if they decline to participate in this study. 
 
If, during this interview, it becomes apparent that the prospective patient / sperm source dyad lacks 
sufficient knowledge and comprehension of the information represented by the elements of informed 
consent to enable them to make an informed and enlightened decision on whether or not to participate 
in the study, they will be excluded from participating in the study. 
 
A copy of signed informed consent document will be obtained and stored in a binder locked in the 
investigators’ office. The consent process, including the name of the individual obtaining consent, will 
be thoroughly documented in the patient/sperm source dyad’s research record. Any alteration to the 
standard consent process (e.g., use of a translator, consent document presented orally, etc.) and the 
justification for such alteration will likewise be documented. 
 
See attached informed consent. 
 
6.3.3 Screening 
All patients/donor dyads potentially undergoing ICSI and meeting inclusion criteria for the study will 
be approached to participate in the study. Screening will be performed by study investigators at NYU 
Langone Reproductive Specialists of NY.  
 
6.3.4 Recruitment, Enrollment and Retention  
Recruitment into the study will be performed by a study investigator at NYU Langone RSOFNY. 
Patient/sperm source dyads are recruited from the study investigators’ practice (RSOFNY) during a 
routine clinical visit. If patient/sperm source dyads desire to enroll in the study, it will be denoted in 
their medical record chart. If they meet eligibility criteria on the day of oocyte retrieval (no evidence of 
severe male factor infertility or surgically derived sperm, at least 6 mature oocytes available for 
randomization, and possibility for ICSI insemination) the study will be performed as per study protocol. 

Study: i21-00021 
Page 21 of 25 
 
In the event that the patient consented to the study but the provided semen sample on the day of 
oocyte retrieval shows severe male factor infertility. The patient/sperm source dyad will then be 
withdrawn from the study since the sample will not qualify for preparation via ZyMot nor density 
gradient. 
 
In the event that the patient/sperm source dyad consented to the study but does not meet the inclusion 
criteria of ICSI insemination or at least 6 mature oocytes available for insemination, insemination will 
occur only with the sperm separated via density gradient, however, the sperm parameters following 
ZyMot and density gradient sperm separation will be recorded. The data on sperm parameters after 
sperm separation via ZyMot and density gradient will still be collected but the patient/sperm source 
dyad will have no further participation in the study. 
 
6.3.5 On Study Visits 
Not applicable. 
 
6.3.6 End of Study and Follow-up 
If the patient/donor dyad is enrolled into the study, the primary and secondary data endpoints will be 
documented as they become available.  
 
No further follow up for those subjects withdrawn from the study will be needed as they will undergo 
ART procedures as per our standard of care.  
 
6.3.7 Removal of subjects  
Following enrollment into the study, the patient/sperm source dyads may withdraw from the study at 
any point prior to insemination of the oocytes. 
 
In the event that the patient/sperm source dyad consented to the study but the provided semen 
sample on the day of oocyte retrieval shows severe male factor infertility as per above criteria, the 
patient/sperm source dyad will be withdrawn from the study since the sample will not qualify for 
preparation via ZyMot nor density gradient. 
 
In the event that the patient/sperm source dyad consented to the study but does not meet the inclusion 
criteria of ICSI insemination or at least 6 mature oocytes available for insemination, insemination will 
occur only with the sperm separated via density gradient, however, the sperm parameters following 
ZyMot and density gradient sperm separation will be recorded. The data on sperm parameters after 
sperm separation via ZyMot and density gradient will still be collected but the patient/sperm source 
dyad will have no further participation in the study. 
 
6.4 Statistical Method 
6.4.1 Statistical Design  
All data will be de-identified and then will be analyzed by a statistician. Descriptive statistics of the 
study sample will be calculated (mean ± standard deviation or median [25th, 75th percentiles] for 
continuous variables; frequency and percent for categorical data. Paired analyses will be performed. 
A result will be considered statistically significant at the p<0.05 level of significance. All analyses will 
be performed using SAS version 9.4 (SAS Institute Inc., Cary, NC). 
 
6.4.2 Sample Size Considerations  
For a two-tailed McNemar’s test with alpha level 0.05, the following relation yields an approximate 
power of 80% to detect a difference d between proportions p1 and p2:n = 16pq (1 - r)/d2 where n is 
the size of the sample (i.e. number of eggs), p = (p1 + p2)/2, q= 1 - p, d = |p2 - p1|, and r is the phi 

Study: i21-00021 
Page 22 of 25 
correlation coefficient1.We will assume a conservative estimate for the correlation r=0.5. Assuming a 
48% good quality blastocyst development rate and a 7% improvement in good quality blastocyst 
development with ZyMoT, we would need a total of 408 oocytes randomized to either density gradient 
or ZyMoT separation, which would equate to 68 patient/sperm source dyads each with ≥6 eggs 
retrieved. 
 
6.4.3 Planned Analyses 
6.4.3.1 Primary Analyses  
Percentage of good quality embryo formation between control and experimental arm will be analyzed 
using McNemar’s test. 
 
6.4.3.2 Secondary Objectives Analyses  
Sperm parameters following ZyMot and density gradient sperm separation, as well as Euploid rates 
in the event that PGT-A was performed following ICSI comparing sperm separation with ZyMot to 
density gradient, will be assessed using the paired t-test, Wilcoxon signed rank test, ora generalized 
linear mixed model (GLMM). A random subject-specific intercept and an unstructured correlation 
structure will be used to account for within-subject correlation between rates between the two groups. 
Data will be reported as least squares means with corresponding 95% confidence intervals. 
 
Pregnancy outcomes will be assessed using McNemar’s test. The difference in good quality 
blastocyst development rate and pregnancy outcomes between the ZyMot and density gradient 
separation groups will also be analyzed separately using a conditional logistic regression model with 
generalized estimating equations to account for both correlation between observations from the same 
patient and the size of the cohort of eggs from each patient. 
 
6.4.3.3 Safety/Pregnancy-related policy 
Not applicable. 
 
6.4.3.4 Analysis of Subject Characteristics 
Subject characteristics will be documented including female age, male age, infertility diagnosis, 
stimulation protocol, medications used, gonadotropin dosage, method of trigger, infertility diagnosis, 
sperm parameters prior to sperm separation, gravity, parity, and previous pregnancy outcomes. 
 
6.4.3.5 Interim Analysis  
An interim analysis will be performed at the midway point of oocyte recruitment. Interim analysis will 
evaluate percentage of good quality embryo formation. Study will be terminated in the event that there 
is a significant decrease in good quality embryo formation in the study arm compared to the control 
arm. 
 
6.4.3.6 Health economic evaluation 
Not applicable.  
 
6.4.3.7 Other 
Not applicable.  
 
6.4.4 Subsets and Covariates  
This study will be randomizing sister oocytes (oocytes from the same patient within the same cycle) 
and thus limiting the impact of confounding variables. 
 
6.4.5 Handling of Missing Data  
 
 
1 Lehr, R. G. 2001. Some practical considerations and a crude formula for estimating sample size for McNemar's 
test. Drug Information Journal, 35: 1227–1233. 

Study: i21-00021 
Page 23 of 25 
Missing data will be maintained as missing in the analysis datasets, unless specified otherwise. If it is 
deemed that imputing data is necessary, the analysis dataset will contain a new variable with the 
imputed value and the original variable value will be maintained as missing. 
 
7 - Trial Administration 
7.1 Ethical Considerations: Informed Consent and HIPAA Authorization  
The study will be conducted in accordance with the ethical principles that have their origin in the 
Declaration of Helsinki. Informed consents will be scanned into the patient's chart and hard copy will 
be stored in a locked file draw/cabinet at NYU Langone RSOFNY. The PI and sub-investigators will 
have access to the locked storage unit.  
 
Data will be stored using codes assigned by the investigators in an excel spreadsheet. The data will 
be collected and stored on REDCap. Only investigators will have access to the samples and data. 
 
There is not any foreseeable possibility that a previously unknown condition (disease, genetic 
disposition, etc.) will be discovered as the result of the study procedure. 
 
Additional information as a result of this study will be added to the patient's medical record and include 
the use of ZyMot to process the sperm specimen and post-processing sperm parameters.  
 
7.2 Institutional Review Board (IRB) Review  
The protocol, informed consent form(s), recruitment materials, and all participant materials will be 
submitted to the IRB for review and approval. Approval of both the protocol and the consent form will 
be obtained prior to enrolling any participants. Any amendment to the protocol will require review and 
approval by the IRB before the changes are implemented to the study. All changes to the consent 
form will be IRB approved. 
 
7.3 Subject Confidentiality  
All data will be stored and analyzed in accordance with NYU's policy on retention of and access to 
research data. Potential identifiable information (patient and cycle ID) will be recorded as a numerical 
number. All research data will be maintained in tangible form and will be retained by the principal 
investigator and other researchers in confidential and secure storage space owned or controlled by 
NYU Langone Health and approved by NYU Langone Health's Medical Center Information 
Technology (MCIT) department. The data will be stored for 6 years after manuscript completion. It will 
be destroyed by deleting it from the storage space. All Research Data containing protected health 
information will be maintained in accordance with New York State laws and regulations. 
 
All hard copies of the informed consents will be stored in a locked file cabinet at the study site and 
only the investigators will have access to the cabinet. 
 
7.4 Deviations/Unanticipated Problems  
Protocol deviations are any noncompliance with the clinical trial protocol. The deviation may be either 
on the part of the participant, the investigator, or the study site staff.  
 
It will be the responsibility of the principal investigator to use continuous vigilance to identify and report 
deviations promptly from the time the investigator becomes aware of the event. All protocol deviations 
will be addressed in study source documents, and reported to the sponsor and to the local IRB per 
the guidelines.  
 
7.5 Data Collection  
Data collection will be the responsibility of the study staff at the site under the supervision of the site 
PI. The investigator will be responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported. Information pertinent to this project will be available in the electronic 

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medical records, clinic records, and a subject datasheet. A study investigator will record demographic 
data and related medical data on the subject datasheet.  
 
All data will be stored and analyzed in accordance with NYU's policy on retention of and access to 
research data. Potential identifiable information (patient and cycle ID) will be recorded as a numerical 
number. All research data will be maintained in tangible form and will be retained by the PI and other 
researchers in confidential and secure storage space owned or controlled by NYU Langone Health 
and approved by NYU Langone Health's Medical Center Information Technology (MCIT) department. 
The data will be stored for 6 years after manuscript completion. It will be destroyed by deleting it from 
the storage space. All Research Data containing protected health information will be maintained in 
accordance with New York State laws and regulations. 
 
All hard copies of the informed consents will be stored in a locked file cabinet at the study site and 
only the investigators will have access to the cabinet. 
 
7.6 Data Quality Assurance 
The study investigators will be responsible for implementing and maintaining quality assurance and 
quality control systems to ensure that the trial is conducted and data are generated, documented 
(recorded), and reported in compliance with the protocol, GCP, and the applicable regulatory 
requirement(s). 
 
The study investigators will be responsible for securing agreement from all involved parties to ensure 
direct access to all trial-related sites, source data/documents, and reports for the purpose of 
monitoring and auditing by the sponsor, and inspection by domestic and foreign regulatory authorities. 
 
Quality control will be applied to each stage of data handling to ensure that all data are reliable and 
have been processed correctly. 
 
Agreements, made by the sponsor with the investigator/institution and any other parties involved with 
the clinical trial, will be in writing, as part of the protocol or in a separate agreement. 
 
7.7 Study Records 
Study records will include patient consents, protocols, electronic medical records, and data related to 
the study. 
 
7.8 Access to Source  
Access to study records will be limited to IRB-approved members of the study team. All data will be 
stored and analyzed in accordance with NYU's policy on retention of and access to research data. 
Potential identifiable information (patient and cycle ID) will be recorded as a numerical number. All 
research data will be maintained in tangible form and will be retained by the PI and other researchers 
in confidential and secure storage space owned or controlled by NYU Langone Health and approved 
by NYU Langone Health's Medical Center Information Technology (MCIT) department. The 
investigator will permit study-related monitoring, audits, and inspections by the IRB/EC, the sponsor, 
government regulatory bodies, and university compliance and quality assurance groups of all study 
related documents (e.g., source documents, regulatory documents, data collection instruments, study 
data etc.). Data may be shared with the sponsor of the study; however, data will be de-identified prior 
to sharing the data. 
 
7.9 Data or Specimen Storage/Security  
All data will be stored and analyzed in accordance with NYU's policy on retention of and access to 
research data. Potential identifiable information (patient and cycle ID) will be recorded as a numerical 
number. All research data will be maintained in tangible form and will be retained by the PI and other 
researchers in confidential and secure storage space owned or controlled by NYU Langone Health 
and approved by NYU Langone Health's Medical Center Information Technology (MCIT) department. 

Study: i21-00021 
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All Research Data containing protected health information will be maintained in accordance with New 
York State laws and regulations. 
 
All hard copies of the informed consents will be stored in a locked file cabinet at the study site and 
only the investigators will have access to the cabinet. 
 
7.10 Retention of Records  
All data will be kept for 6 years following completion of the study. If permission is needed to move or 
destroy the records, it will be the PI's responsibility.  
 
7.11 Study Monitoring  
Halfway through recruitment of sample size, preliminary results will be analyzed to by the principal 
investigator and co-investigators. Information related to the study will be periodically evaluated for 
accuracy by one or more of the study investigators.  
 
7.12 Data Safety Monitoring Plan 
Halfway through recruitment of sample size, preliminary results will be analyzed by the principal 
investigator and co-investigators. If the primary endpoint is adversely affected by study arm, the study 
will be terminated. The primary endpoint will be percentage of good quality embryos comparing those 
inseminated with density processed sperm and ZyMot processed sperm. In the event that there is a 
statistically significant (p<0.05) percentage of higher quality embryo formation in the embryos created 
via sperm inseminated with density processed sperm compared to ZyMot processed sperm, the study 
will be terminated. Preliminary analysis of the results will be communicated promptly to the IRB. 
 
7.13 Study Modification 
If at any points study modifications are to be made, the protocol will be updated and changes 
submitted to the IRB. Changes to the protocol will be implemented once approved by the IRB. 
 
7.14 Study Discontinuation 
In the event that preliminary analysis reveals significantly reduced primary outcome in the 
experimental arm, the study will be discontinued.  
 
7.15 Study Completion  
Study will be completed when 408 oocytes are randomized which is anticipated to be in approximately 
1-2 years from study commencement.  
  
7.16 Conflict of Interest Policy 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, or financial 
gain greater than the minimum allowable by their institution, etc.) will have the conflict reviewed by 
the NYU Langone Conflict of Interest Management Unit (CIMU) with a Committee-sanctioned conflict 
management plan that has been reviewed and approved by the study sponsor prior to participation in 
this study. All NYULMC investigators will follow the applicable conflict of interest policies. 
 
7.17 Funding Source 
The study will be funded by DxNow Inc, manufacturer of the ZyMot device. 
 
7.18 Publication Plan 
Publication procedures and authorship will be addressed in the Clinical Trials Agreement for this 
study.