Number eight Sarah Hunter and flanker Marlie Packer will co-captain England as they begin the defence of their Women's Six Nations title this month.

Scrum-half Natasha Hunt and number eight Sarah Beckett return after being left out of England's World Cup squad.

Nine uncapped players are also included in the 42-player squad.

Saracens pair May Campbell and Kelsey Clifford are selected alongside Sale Sharks fly-half Elizabeth Duffy and Exeter Chiefs centre Nancy McGillivray, who have both featured for England Under-20s.

England begin their campaign against Scotland in Newcastle on Saturday, 25 March (16:45 GMT).

Pregnant lock Abbie Ward and regular centre and vice-captain Emily Scarratt, who is injured, are unavailable for selection but will remain with the group and assist the coaching set-up.

"There are a number of new faces in this squad and while we are missing experienced players, this is a great opportunity for these younger players and we're looking forward to working with them," said Middleton.

"There is always a regeneration period after any World Cup and this one is no different.

"Sarah has made it clear she will not be involved in the 2025 World Cup as a player, so it's essential we look to continue to grow our leadership quality and depth.

"Marlie is already an integral part of our leadership group, so this is a natural progression for her to co-captain the side and continue to develop her leadership skills."

Middleton became head coach in 2015 and has led England to five Six Nations titles and four Grand Slams.

The search for his successor continues, and he says coaching is an area of the women's game which is one of the "next big pillars" to put in place.

He added: "Both Abbie Ward and Emily Scarratt expressed a wish to support our Six Nations campaign despite their playing absence.

"Both are an integral part of our strategy and leadership group and in my opinion, have great coaching potential in terms of their knowledge and their delivery.

"Coaching is an area of the women's game we all recognise as one of the next big pillars to put in place, so it stands to reason that we jump on this opportunity to support both in an area they have expressed a wish to develop."

England squad

Forwards: Zoe Aldcroft, Sarah Beckett, Sarah Bern, May Campbell, Bryony Cleall, Poppy Cleall, Kelsey Clifford, Amy Cokayne, Liz Crake, Lark Davies, Rosie Galligan, Detysha Harper, Sarah Hunter, Sadia Kabeya, Poppy Leitch, Alex Matthews, Maud Muir, Cath O'Donnell, Marlie Packer, Connie Powell, Emily Robinson, Hannah Sims, Morwenna Talling

Backs: Holly Aitchison, Jess Breach, Abby Dow, Elizabeth Duffy, Tatyana Heard, Natasha Hunt, Leanne Infante, Ellie Kildunne, Claudia MacDonald, Nancy McGillivray, Sarah McKenna, Lucy Packer, Amber Reed, Helena Rowland, Ellie Rugman, Emma Sing, Lydia Thompson, Lagi Tuima, Ella Wyrwas

Analysis

BBC rugby reporter Sara Orchard

There are some big gaps in England's armoury, particularly at fly-half and prop. How brave Middleton will be to hand out a raft of new caps during the tournament could be better determined by the will of forwards coach Louis Deacon.

He remains the only senior coach who will be staying on the Red Roses coaching ticket beyond the Six Nations. With only a handful of tournaments before England host the 2025 World Cup, every Test matters.

Regular captain Sarah Hunter is expected to announce her retirement imminently, meaning Saracens' Marlie Packer has the opportunity to cement herself as front and centre of the Red Roses' ambitions.