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One group stage spot is on offer to the winner of the two-legged affair, with the second leg set to take place at Stadio Tre Fontane on Thursday 29 September (14:30 local time).
Ahead of the first leg on Wednesday evening (kick-off: 18:30 CEST), we've rounded up everything you need to know...
General Statistics
Roma have reached this stage of the competition - in their inaugural appearance - after beating Glasgow City (3-1) and Paris FC (0-0 and penalties) in the first phase.
Their last Serie A match was a narrow 1-0 away defeat to Juventus, who are also in Champions League action this week.
That match against Juventus was the first time in 19 games that the side had failed to get on the scoresheet, and the last time they had lost a league match.
Focus on the Opposition
Sparta Prague were eliminated at this stage of last season's Champions League, losing against Koge (1-0 in the first leg and 2-0 in the second).
The season prior to that, however, the Czech side reached the last-16 of the competition - where they were knocked out by Paris Saint-Germain.
The side are currently joint top of their domestic league, with 12 points from 12 so far.
Roma Players in focus
Benedetta Glionna is the club's top goalscorer in the Champions League to date, having scored twice in the debut win over Glasgow.
Emilie Haavi scored the decisive penalty to beat Paris FC in Scotland and reach this stage; the Norwegian winger has played 441 minutes this season, behind only Moeka Minami (480) and Manuela Giugliano (455).
Minami, meanwhile, was first for passes attempted (85), passes completed (78) and touches of the ball (93) during the last round of Serie A matches.
The next appearances for both Camelia Ceasar and Paloma Lazaro will be their 60th in all competitions for the Giallorosse.
Sparta Prague in focus
American forward Amber Tripp is the club's top goalscorer so far this season - she has four goals so far, including a hat-trick on the opening day of the season.
Eliska Sonntagova (3) and Lucie Martinkova (3) have also been significant goal threats so far this season in the Czech league.
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