Methods and test kits for determining male fertility status

A method comprising:

• a. obtaining a first portion of a portion of a sperm sample from a male that has been exposed to in vitro capacitating conditions, fixed in a fixative, and stained with a labeling molecule for GM1 localization patterns, wherein the labeling molecule has a detectable label;
• b. identifying a plurality of GM1 labeled localization patterns for the labeled fixed in vitro capacitated sperm sample, said plurality of GM1 localization patterns comprising an apical acrosome (AA) GM1 localization pattern, an acrosomal plasma membrane (APM) GM1 localization pattern, a Lined-Cell GM1 localization pattern and all other labeled GM1 localization patterns;
• c. assigning the AA GM1 localization pattern and the APM GM1 localization pattern to a capacitated state;
• d. assigning the Lined-Cell GM1 localization pattern and all other labeled GM1 localization patterns to a non-capacitated state;
• and
• e. characterizing a fertility status of the male by applying one or more pre-trained fertility classifiers to data obtained from the sperm sample, wherein the data obtained from the sperm sample comprises a ratio between (i) a combination of the AA GM1 localization pattern and APM GM1 localization pattern and (ii) a combination of the GM1 labeled localization patterns (e.g., a ratio of sperm displaying a capacitated state to a total number of assigned sperm).

A method comprising the steps of:

• a. obtaining a sperm sample, wherein at least a portion of the sperm sample has been exposed to in vitro capacitating conditions to obtain an in vitro capacitated sperm, has been exposed to a fixative, and has been stained for GM1, thereby forming a labeled fixed in vitro capacitated sperm sample;
• b. determining a Cap-Score of the labeled fixed in vitro capacitated sperm sample based on one or more GM1 labeled localization patterns, said GM1 labeled localization patterns being an apical acrosome (AA) GM1 localization pattern, a post-acrosomal plasma membrane (APM) GM1 localization pattern, a Lined-Cell GM1 localization pattern and all other labeled GM1 localization patterns;
• and
• c. characterizing a fertility status of the male by applying one or more pre-trained fertility classifiers to data obtained from the sperm sample, wherein the data comprises a ratio between (i) a combination of the AA GM1 localization pattern and the APM GM1 localization pattern and (ii) a combination of all the GM1 labeled localization patterns (e.g., a ratio of sperm displaying a capacitated state to a total number of assigned sperm).

A method, comprising:

• a. characterizing a fertility status of a male by applying one or more pre-trained fertility classifiers to data obtained from a sperm sample from the male, wherein the data comprises a ratio between (i) a combination of apical acrosome (AA) GM1 localization patterns and acrosomal plasma membrane (APM) GM1 localization patterns and (ii) a combination all GM1 labeled localization patterns in a treated portion of the sperm sample,
• b. wherein the ratio between (i) the combination of the AA GM1 localization patterns and APM GM1 localization patterns and (ii) the combination of all GM1 labeled localization patterns is determined by:
• a. exposing, in vitro, a portion of the sperm sample from the male to capacitating conditions, thereby forming a capacitated sperm sample;
• b. fixing the capacitated sperm sample with a fixative, thereby forming a fixed in vitro capacitated sperm sample;
• c. treating the fixed in vitro capacitated sperm sample with a labeling molecule for GM1 localization patterns, wherein the labeling molecule has a detectable label, thereby forming a labeled fixed in vitro capacitated sperm sample;
• d. identifying a plurality of GM1 labeled localization patterns for the labeled fixed in vitro capacitated sperm sample, said plurality of GM1 labeled localization patterns comprising an AA GM1 localization pattern, an APM GM1 localization pattern, a Lined-Cell GM1 localization pattern and all other labeled GM1 localization patterns;
• e. assigning the AA GM1 localization pattern and the APM GM1 localization pattern to a capacitated state;
• f. assigning the Lined-Cell GM1 localization pattern and all other labeled GM1 localization patterns to a non-capacitated state;
• and
• g. comparing (i) the combination of the AA GM1 localization pattern and APM GM1 localization pattern to (ii) the combination of all the GM1 labeled localization patterns.

A system for training a fertility classifier for characterizing a fertility status of a male, the system comprising:

• a. at least one processor and memory addressable by the at least one processor, the memory storing at least one program for execution by the at least one processor, the at least one program comprising instructions for:
• i. obtaining a training set that comprises data from sperm samples from a plurality of males associated with a known outcome of an attempt at assisted reproduction (e.g., intra-uterine insemination (IUI)), wherein the data from each respective semen sample comprises a ratio between (x) a combination of the AA GM1 localization pattern and APM GM1 localization pattern and (y) the combination of all the GM1 labeled localization patterns of sperm in the respective semen sample (e.g., a ratio of sperm displaying a capacitated state to a total number of assigned sperm);
• and
• ii. training one or more fertility classifiers based on at least a correspondence between the outcome of the assisted reproduction attempt and the corresponding ratio between (i) a combination of the AA GM1 localization pattern and APM GM1 localization pattern and (ii) the combination of all the GM1 labeled localization patterns of sperm in each respective semen sample.