normal gross morphology) from the patient's sample, this pro-

cess does not ensure the genetic integrity of the sperm, and

hence, the resultant embryo.

As a subpopulation, men with normal semen parameters

who are partners in a couple with recurrent pregnancy loss

(RPL) or unexplained recurrent IVF failure are commonly

overlooked. Sperm aneuploidies in these normozoospermic

men could represent a signi

cant, but clinically under-

appreciated, cause of infertility. In this context, cytogenetic

analysis of sperm using

ﬂ

uorescence in situ hybridization

(FISH) can help evaluate potential causes of recurrent preg-

nancy loss or recurrent IVF failure. In this study, we investi-

gated the incidence of autosome and sex chromosome

aneuploidies in the sperm of men who are partners in couples

with RPL and IVF failure.

MATERIALS AND METHODS

A total of 140 male partners of couples who presented with

RPL were analyzed. We de

ned RPL as recurrent miscarriage

and/or the inability to achieve a pregnancy with IVF/ICSI. We

included men who had [1] sperm aneuploidy testing with

FISH, [2] at least two semen analyses on 2 separate days, [3]

at least one strict morphology (assessed with Kruger criteria),

and [4] a sperm DNA fragmentation assay (assessed with

TUNEL).

Sperm density and motility were averaged from the

semen parameters available. Abnormal sperm density was

ned as

sperm/mL and abnormal motility as

40%, according to the World Health Organization 2010

guidelines. A total of 140 semen samples from

ve normozoo-

spermic men were used as controls for comparison. We

excluded men with known causes of infertility, such as Klein-

felter syndrome and Y-chromosome microdeletions, and men

who did not have at least two semen analyses for evaluation.

The institutional review board at the Baylor College of Medi-

cine approved the study.

FISH Assay

Semen samples obtained for FISH testing were centrifuged for

10 minutes at 1,900 rpm to separate seminal

ﬂ

uid from cells

(within 24 hours). The supernatant was removed, 5 mM KCl

was added, and the sample was incubated for 25 minutes at

C. Carnoy

x (three parts methanol plus one part acetic

acid) was added, and the sample was recentrifuged. The step

was repeated until the pellet was white and the supernatant

clear. Approximately 5 mL of the sample was

xed on a slide

and the slide stored at

C before processing for FISH.

Three-color FISH was performed on ejaculated sperm, to

help de

ne the numerical abnormalities in chromosome 18

and the sex chromosomes (X and Y). Two-color FISH was

used to detect chromosome abnormalities in chromosomes

13 and 21. A minimum of 20,000 sperm were scored for

each man. The VYSIS AneuVysion DNA Probe Kit (catalog

no. 35-161075) was used (incorporating CEP probes for chro-

mosomes 13 and 21 and LSI probes for chromosome X, Y and

chromosome 18).

Scoring of sperm nuclei was performed under an Olympus

BX51

ﬂ

uorescent microscope using an aqua

lter (C43494

excitation peak, 433 nm; emission peak, 480 nm) for

chromosome 18, a green

lter (U-MNB2 excitation peak,

330

–

385 nm; emission peak, 420 nm) for chromosomes X

and 13, a red

lter (U-MGN2 excitation peak, 530

–

550 nm;

emission peak, 590 nm) for the Y and 21 chromosome, and

lter U-M@U2 for 6-diamino-2-phenylindole (excitation,

330

–

385 nm; emission peak, 420 nm). Images were captured

using Image Pro software.

Sperm were scored as disomic if the same-colored signals

were of similar intensity, size, and shape, and if both signals

were clearly visible within the sperm. In addition, positively

scored cells had a clearly de

ned border and were not overlap-

ping. One technician speci

cally trained in FISH analysis per-

formed all tests described. We de

ned an abnormal FISH

parameter as having an aneuploidy percentage

2 SDs from

the mean of our normozoospermic controls. Results can be

standardized in our patient samples given that the proportion

of aneuploidy in our control population is similar to that re-

ported in the published literature on aneuploidy in the general

population

(6)

. We tested normality of the distribution using a

quantile-quantile plot (QQ plot) and Shapiro Wilk test and as-

certained that the data were normally distributed.

Statistical Analysis

Statistical analysis was performed using SPSS version 22

(IBM). Means were compared using an independent-sample

TABLE 1

Baseline characteristics of men with RPL.

Characteristic

Men with RPL

Control

value

140

–

Age (y)

30.5

23.5

.335

Sperm density (

/mL)

37.5

116.8

.001

Sperm motility (%)

46.7

62.2

.001

Sex disomy (%)

1.04

0.38

.015

18 disomy (%)

0.18

0.03

.001

13/21 disomy (%)

0.26

0.08

.002

Men with sex chromosome disomy, % (n)

52.9 (74/140)

2.9 (4/140)

.001

Men with chromosome 18 disomy, % (n)

36.4 (51/140)

29.3 (41/140)

.252

Men with chromosomes 13/21 disomy, % (n)

50.0 (70/140)

52.9 (74/140)

.720

Ramasamy. Sperm FISH and recurrent pregnancy loss. Fertil Steril 2015.

VOL. 103 NO. 4 / APRIL 2015

907

Fertility and Sterility®